Structural and functional characterization of a single-chain peptide-MHC molecule that modulates both naive and activated CD8+ T cells.
ABSTRACT Peptide-MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8(+) T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8(+) T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scK(d).IGRP) by using the class I MHC molecule H-2K(d) and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP(206-214)), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scK(d).IGRP tetramers bound specifically to cognate CD8(+) T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-γ response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8(+) T cells makes them a potential intervention strategy in early and late stages of disease.
- [show abstract] [hide abstract]
ABSTRACT: Upon Ag exposure, most memory T cells undergo restimulation-induced cell death. In this article, we describe a novel synthetic agonist, an N-terminal extended decamer peptide expressed as a single-chain trimer, the amino-terminal extended peptide MHC class I single-chain trimer (AT-SCT), which preferentially promotes the growth of memory human CD8(+) T cells with minimal restimulation-induced cell death. Using CMV pp65 and melanoma gp100 Ags, we observe the in vitro numerical expansion of a clonally diverse polyfunctional population of Ag-specific CD8(+) T cells from healthy individuals and vaccinated melanoma patients, respectively. Memory CD8(+) T cells stimulated with AT-SCT presented on MHC class I/II-null cells show reduced cytokine production, slower kinetics of TCR downregulation, and decreased cell death compared with native nonamer MHC class I single-chain trimer (SCT)-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT- and SCT-activated T cells. Probing of SCT and AT-SCT peptide-MHC complexes using fluorochrome-conjugated TCR multimers suggests that nonamer- and decamer-linked peptides may be anchored differently to the HLA-A2 peptide-binding groove. Our findings demonstrate that modified peptide-MHC structures, such as AT-SCT, can be engineered as T cell agonists to promote the growth and expansion of memory human CD8(+) T cells.The Journal of Immunology 05/2012; 188(12):5839-49. · 5.52 Impact Factor
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ABSTRACT: Classical major histocompatibility complex (MHC) class I and II molecules present peptides to cognate T-cell receptors on the surface of T lymphocytes. The specificity with which T cells recognize peptide-MHC (pMHC) complexes has allowed for the utilization of recombinant, multimeric pMHC ligands for the study of minute antigen-specific T-cell populations. In type 1 diabetes (T1D), CD8+ cytotoxic T lymphocytes, in conjunction with CD4+ T helper cells, destroy the insulin-producing β cells within the pancreatic islets of Langerhans. Due to the importance of T cells in the progression of T1D, the ability to monitor and therapeutically target diabetogenic clonotypes of T cells provides a critical tool that could result in the amelioration of the disease. By administering pMHC multimers coupled to fluorophores, nanoparticles, or toxic moieties, researchers have demonstrated the ability to enumerate, track, and delete diabetogenic T-cell clonotypes that are, at least in part, responsible for insulitis; some studies even delay or prevent diabetes onset in the murine model of T1D. This paper will provide a brief overview of pMHC multimer usage in defining the role T-cell subsets play in T1D etiology and the therapeutic potential of pMHC for antigen-specific identification and modulation of diabetogenic T cells.Clinical and Developmental Immunology 01/2012; 2012:380289. · 3.06 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: CD8(+) T cells specific for islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) have been implicated in type 1 diabetes in both humans and non-obese diabetic (NOD) mice, in which T cells specific for IGRP206-214 are highly prevalent. We sought to manipulate these pathogenic T cells by exploiting the ability of steady-state dendritic cells (DCs) to present antigens in a tolerogenic manner. The endocytic receptor DEC-205 was utilized to deliver an IGRP206-214 mimotope to DCs in NOD mice, and the impact of this delivery on a polyclonal population of endogenous islet-reactive cognate T cells was determined. Assessment of islet-infiltrating CD8(+) T cells showed a decrease in the percentage, and the absolute number, of endogenous IGRP206-214-specific T cells when the mimotope was delivered to DCs, compared with delivery of a specificity control. Employing an adoptive transfer system, deletion of CD8(+) T cells as a result of DEC-205-mediated antigen targeting was found to occur independently of programmed death-1 (PD-1) and its ligand (PD-L1), both often implicated in the regulation of peripheral T-cell tolerance. Given its promise for the manipulation of self-reactive polyclonal T cells demonstrated here, the distinctive characteristics of this antigen delivery system will be important to appreciate as its potential as an intervention for autoimmune diseases continues to be investigated.International Immunology 09/2013; · 3.14 Impact Factor
Structural and functional characterization
of a single-chain peptide–MHC molecule that
modulates both naive and activated CD8+T cells
Dibyendu Samantaa,1, Gayatri Mukherjeea,1, Udupi A. Ramagopalb, Rodolfo J. Chaparroa, Stanley G. Nathensona,c,2,
Teresa P. DiLorenzoa,d,2, and Steven C. Almob,e,2
Departments ofaMicrobiology and Immunology,bBiochemistry,cCell Biology,dMedicine/Division of Endocrinology, andePhysiology and Biophysics, Albert
Einstein College of Medicine, Bronx, NY 10461
Contributed by Stanley G. Nathenson, July 7, 2011 (sent for review May 31, 2011)
Peptide–MHC (pMHC) multimers, in addition to being tools for
tracking and quantifying antigen-specific T cells, can mediate
downstream signaling after T-cell receptor engagement. In the
absence of costimulation, this can lead to anergy or apoptosis of
cognate T cells, a property that could be exploited in the setting of
autoimmune disease. Most studies with class I pMHC multimers
used noncovalently linked peptides, which can allow unwanted
CD8+T-cell activation as a result of peptide transfer to cellular
MHC molecules. To circumvent this problem, and given the role
of self-reactive CD8+T cells in the development of type 1 diabetes,
we designed a single-chain pMHC complex (scKd.IGRP) by using
the class I MHC molecule H-2Kdand a covalently linked peptide
derived from islet-specific glucose-6-phosphatase catalytic sub-
unit-related protein (IGRP206–214), a well established autoantigen
in NOD mice. X-ray diffraction studies revealed that the peptide is
presented in the groove of the MHC molecule in canonical fashion,
and it was also demonstrated that scKd.IGRP tetramers bound spe-
cifically to cognate CD8+T cells. Tetramer binding induced death of
naive T cells and in vitro- and in vivo-differentiated cytotoxic
T lymphocytes, and tetramer-treated cytotoxic T lymphocytes
showed a diminished IFN-γ response to antigen stimulation. Tet-
ramer accessibility to disease-relevant T cells in vivo was also dem-
onstrated. Our study suggests the potential of single-chain pMHC
tetramers as possible therapeutic agents in autoimmune disease.
Their ability to affect the fate of naive and activated CD8+T cells
makes them a potential intervention strategy in early and late
stages of disease.
MHC molecules, and this recognition can lead to the demise of
the cell displaying the cognate peptide–MHC (pMHC) complex.
As a result, CD8+T cells are important pathogenic effectors in
a number of autoimmune diseases, including type 1 diabetes (1).
The development of strategies to interfere with their function
offers new therapeutic opportunities. Treatment of CTLs with
multimers of pMHC complexes has shown promise in inhibiting
CTL-mediated cytotoxicity (2–5). For example, pMHC multi-
mers constructed with short flexible linkers cause rapid death of
peptide-specific CTLs (3), whereas those with long rigid linkers
inhibit CTL-mediated cytotoxicity by interfering with integrin-
mediated CTL adhesion (2). In addition, dimeric Ig fusions of
pMHC complexes have been shown to inhibit lysis of target cells
by alloreactive CTLs (4, 5).
We reasoned that, in addition to their inhibition of already
differentiated CTLs (2–5), pMHC multimers should also be ef-
fective against naive T cells, as they would present antigen in the
absence of a second costimulatory signal and would be predicted
to drive the T cells to apoptosis or anergy (6–9). This is a pro-
foundly unexplored area, perhaps because of the early un-
expected finding that pMHC tetramers could instead activate
naive CD8+T cells (10). This behavior was subsequently found to
result from the release of the peptide from the tetramers and its
transfer to MHC molecules on T cells, which then acted as
antigen-presenting cells capable of activating their naive coun-
D8+cytotoxic T lymphocytes (CTLs) use their T-cell re-
ceptors (TCRs) to recognize peptides presented by class I
terparts (11, 12). Thus, the activity of pMHC multimers against
CD8+T cells, both naive and antigen-experienced, requires
reevaluation with the use of pMHC complexes in which the
peptide is rendered nonexchangeable by virtue of covalent
linkage to the complex (13, 14).
To this end, we used a disease-relevant model system con-
sisting of autoreactive CD8+8.3 T cells. The 8.3 T-cell clone was
originally isolated from the pancreatic islets of a nonobese di-
abetic (NOD) mouse (15), a model system for type 1 diabetes in
which CD8+T cells have an important pathogenic role (16).
The 8.3 T-cell clone is specific for the peptide composed of
residues 206 to 214 of islet-specific glucose-6-phosphatase cata-
lytic subunit-related protein (IGRP206–214) presented by H-2Kd
(17), and its pathogenicity has been demonstrated by adoptive
transfer studies (15) and the accelerated disease that occurs in
NOD mice that transgenically express the 8.3 TCR (18). T cells
specific for IGRP206–214represent a prevalent population in the
islets of NOD mice (17, 19, 20), and the monitoring of their
numbers in the blood can be used to predict disease (20). IGRP
epitopes have also been found to be targeted by CD8+T cells in
human type 1 diabetes (21–23).
We used 8.3 T cells to investigate whether a single multimeric
pMHC reagent could be developed that would inactivate or
eradicate both CTLs and naive CD8+T cells. We designed a
single-chain pMHC complex in which IGRP206–214is covalently
attached to β2-microglobulin (β2m), which itself is covalently
linked to the heavy chain of H-2Kd. X-ray diffraction analysis of
the single-chain H-2Kd/IGRP206–214(scKd.IGRP) demonstrated
that the covalently linked peptide is presented in the canonical
binding groove of the MHC molecule in a fashion that would
support productive TCR engagement. Tetramers of scKd.IGRP
exhibit high-specificity binding for the cognate 8.3 TCR. Most
importantly, scKd.IGRP tetramers specifically induce apoptosis
of naive CD8+8.3 T cells, as well as of in vitro-generated CTLs
and islet-infiltrating CTLs naturally differentiated in vivo. The
tetramers also gain access to splenic and pancreatic T cells when
administered in vivo. These characteristics support further ex-
ploration of the therapeutic potential of single-chain pMHC
tetramers for type 1 diabetes and other conditions in which
CD8+T cells contribute to the pathogenic process.
Author contributions: D.S., G.M., S.G.N., T.P.D., and S.C.A. designed research; D.S., G.M.,
U.A.R., and R.J.C. performed research; D.S. contributed new reagents/analytic tools; D.S.,
G.M., U.A.R., and T.P.D. analyzed data; and D.S., G.M., S.G.N., T.P.D., and S.C.A. wrote the
The authors declare no conflict of interest.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID code 3NWM).
1D.S. and G.M. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org, teresa.
email@example.com, or firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 16, 2011
| vol. 108
| no. 33www.pnas.org/cgi/doi/10.1073/pnas.1110971108
Design and Biochemical Characterization of scKd.IGRP. To circum-
vent the complications associated with peptide transfer to cel-
lular MHC molecules (11, 12), we designed the single-chain
construct scKd.IGRP, which contained the heavy chain of H-2Kd,
β2m, and the target peptide, following a strategy similar to that
reported for a single-chain construct of H-2Kbpresenting
a peptide derived from ovalbumin (13, 14). Specifically, the
C terminus of IGRP206–214(VYLKTNVFL) was fused to the
N terminus of β2m with a GGGAS(G4S)2linker and the C ter-
minus of β2m was fused to the N terminus of the H-2Kdheavy
chain with a (G4S)4linker (Fig. 1A).
The scKd.IGRP construct was expressed in Escherichia coli as
inclusion bodies that were refolded and purified in milligram
quantities (Fig. S1A). The refolded material exhibited excellent
solution properties and was monodisperse as demonstrated
by analytical size-exclusion chromatography (Fig. S1C), and
exhibited a well resolved single tight band on a native poly-
acrylamide gel (Fig. S1B). A control single-chain H-2Kdmole-
cule presenting the tumor-derived peptide KYQAVTTTL (24),
which is not recognized by 8.3 T cells, was prepared in an
identical fashion and designated scKd.TUM (Fig. S2).
Anchoring of IGRP206–214in H-2KdGroove and Implications for TCR
Recognition. The bacterially expressed and refolded scKd.IGRP
protein crystallized in the monoclinic space group P21with one
molecule per asymmetric unit. Initial phase estimates were
obtained by molecular replacement using a conventional H-2Kd
complex (Protein Data Bank ID 2FWO) (25) incorporating the
influenza virus-derived peptide TYQRTRALV (FLU). After
initial refinement, interpretable electron density was observed
for the IGRP206–214peptide and the two linker glycine residues
immediately C-terminal to the peptide (Fig. 1B). The final elec-
tron density map was of excellent quality, with no ambiguities
observed for the main-chain or side-chain atoms of IGRP206–214,
except for the phenylalanine present at position 8 of the peptide
(Fig. 1C and Fig. S3). Although the electron density of that
phenylalanine side chain was weak, electrospray ionization
Fourier transform MS revealed a mass of 49,463.6 Da, which is
consistent with the predicted molecular weight of 49,463.1 Da,
including the phenylalanine at position 8 (Fig. S4). The phe-
nylalanine side chain was modeled on the basis of weak density
and occupies one of the most favored rotamer positions. Dif-
fraction data to 2.7 Å resolution were used for refinement,
heavy chain are depicted in green, red, and blue, respectively. The same color scheme is maintained in B. At the C terminus of the heavy chain, the biotinylation
site is represented as a thin white box. (B) Ribbon diagram of scKd.IGRP. The IGRP peptide is rendered as a ball-and-stick model and the atoms are colored as
follows: carbon, green; nitrogen, blue; and oxygen, red. The complex is oriented with the IGRP N terminus on the top left and the membrane proximal α3
domain at the bottom. The linkers between the IGRP peptide and β2m and between β2m and the heavy chain are represented as dotted lines. The two linker
glycines immediately C-terminal to the peptide are shown in black. (C) Experimental electron density and overall conformation of the IGRP peptide. The Fo-Fc
map contoured at the 2σ level is shown around the peptide. The membrane distal peptide-binding platform of H-2Kdis depicted as a blue ribbon and the IGRP
peptide is displayed as a ball-and-stick model. The N terminus of the peptide is at the top of the figure. (D) Ribbon representation of the heavy chain and ball-
and-stick representation of the IGRP peptide emphasizes the orientation of the side chains of the IGRP peptide (labeled). (E) Superposition of scKd.IGRP onto the
structure of H-2Kd/FLU (2FWO), which reflects a very high degree of similarity between these two complexes. The CαRMSD calculated over all of the residues of
β2m and the heavy chain is 1.08 Å. Both structures are shown as ribbon representations, with the H-2Kd/FLU complex in cyan.
Single-chain pMHC design and structural features. (A) Schematic representation of the scKd.IGRP construct is shown. The IGRP peptide, β2m, and the
Samanta et al.PNAS
| August 16, 2011
| vol. 108
| no. 33
resulting in a final atomic model with Rworkand Rfreeof 20.8%
and 28.4%, respectively, and good stereochemistry (Table S1).
Despite the importance of H-2Kd/IGRP206–214as a target of
pathogenic CD8+T cells in type 1 diabetes in NOD mice (17, 19,
20), the structure of this pMHC complex has not been reported
previously to our knowledge. We found that IGRP206–214
(VYLKTNVFL) is presented in the H-2Kdgroove between the
α1and α2helices and on top of the β-sheet platform in canonical
fashion (Fig. 1B and C) (26). The overall conformation of the
IGRP peptide is shown in Fig. 1D and Fig. S5. The binding of the
IGRP peptide is associated with complete or partial burial of
TyrP2, ThrP5, and LeuP9in the H-2Kdgroove (Fig. 1D and Table
S2). Tyrosine is found at the P2 position of nearly all naturally
processed H-2Kd–binding nonameric peptides (25). Amino acids
present at the C-terminal P9 position are also highly conserved,
with a preference for Ile, Leu, and Val (25). Our structural data
suggest that TyrP2and LeuP9are indeed important residues for
anchoring IGRP206–214in the H-2Kdgroove. Our structural data
also suggest that LysP4, AsnP6, ValP7, and PheP8are the most
likely to be involved in contacting residues of the cognate TCRs,
as the side chains of these residues protrude from the groove and
are accessible for TCR recognition (Fig. 1D and Table S2). This
is consistent with the identification of LysP4and PheP8as major
8.3 TCR contact residues (27), and the finding that alteration of
P7 in the IGRP206–214mimotope peptide NRP (KYNKANWFL)
to Ala or Val endows the resulting NRP-A7 and NRP-V7 pep-
tides with superagonist activity (28).
Structural alignment of scKd.IGRP with the native (i.e., non-
covalently linked) H-2Kd/FLU (25) highlights a high degree of
similarity between these two complexes (Fig. 1E), with an rmsd
of 1.06 Å calculated over all Cαatoms from the β2m domain, the
heavy chain, and the peptide (0.47 Å between the β2m domains
and 1.08 Å between the heavy chains). Overall, the structural
organization of the complex and the canonical mode of peptide
binding are conserved in the covalently linked pMHC complex.
These results further suggest that the single-chain pMHC com-
plex presents peptide and contacts its cognate TCR in a canon-
Tetramerization of scKd.IGRP. Because of the relatively weak bind-
generated scKd.IGRP tetramers with enhanced avidity to assess
the functional activity of the single-chain pMHC complex. The
tetramers were constructedby using standard procedures based on
the high-affinity biotin–streptavidin interaction. The scKd.IGRP
highly homogeneous peaks on size-exclusion chromatography
(Fig. 2A and Fig. S2B). A potential challenge associated with the
production of such tetramers is the generation of heterogeneous
anistic interpretations (29). Our preparations consisted nearly ex-
clusively of tetrameric complexes (Fig. 2A and Fig. S2B).
Specific Binding of scKd.IGRP Tetramers to CD8+T Cells Bearing
a Cognate TCR. To assess the binding capacity of the scKd.IGRP
for the cognate 8.3 TCR, scKd.IGRP was tetramerized by using
phycoerythrin (PE)-labeled streptavidin, and its binding to
splenocytes from 8.3 TCR-transgenic NOD mice was assessed by
flow cytometry. Tetramers of scKd.IGRP bound to nearly all the
CD8+T cells from these mice, whereas PE-conjugated tetramers
of scKd.TUM did not bind (Fig. 2B). This behavior indicates that
the scKd.IGRP complex adopts a conformation in which the
covalently linked antigenic peptide is properly presented for
specific recognition by the cognate 8.3 T cells. The specificity of
the binding was further substantiated by the lack of interaction
between scKd.IGRP tetramers and AI4 T cells (Fig. 2B), which
recognize an autoantigenic peptide in the context of H-2Db(19).
scKd.IGRP Tetramers Do Not Activate Cognate Naive CD8+T Cells and
Instead Drive Them to Apoptosis. To examine the biological activity
of scKd.IGRP tetramers on naive T cells, splenocytes isolated
from nondiabetic 8.3 TCR-transgenic NOD mice were treated
with scKd.IGRP or scKd.TUM tetramers for 3 h. Splenic CD8+
T cells from these mice were previously demonstrated to be
largely naive (18). We confirmed this observation on the basis of
high CD62L expression, which was insensitive to tetramer treat-
ment (Fig. 3A), showing that the tetramer binding did not activate
the CD8+T cells. However, staining with Annexin V–FITC
demonstrated that the scKd.IGRP tetramers induced phosphati-
dylserine externalization, a marker of apoptosis, in nearly 20% of
cognate CD8+8.3 T cells, whereas the irrelevant scKd.TUM
tetramers did not have this effect (Fig. 3B). In contrast, the scKd.
IGRP tetramers were unable to induce apoptosis of noncognate
CD8+AI4 T cells (Fig. 3C).
Death of in Vitro- and in Vivo-Generated CTLs upon Treatment with
scKd.IGRP Tetramers. Peptide–MHC class I multimers, which are
widely used as cell-surface staining reagents, have been reported
to inhibit CTL activity and induce apoptosis of differentiated
CD8+T cells (2–5). However, single-chain pMHC multimers
were not examined in these earlier studies. To explore the effect
of the scKd.IGRP tetramers on CTLs, we first generated CTLs
in vitro by using splenocytes from 8.3 TCR-transgenic NOD
mice. CTLs were then treated with the single-chain tetramers for
3 h and stained with Annexin V–FITC to assess apoptosis in-
duction. It was found that, as with naive cells, the CTLs were also
driven to death specifically by the scKd.IGRP tetramers, whereas
the noncognate scKd.TUM tetramers had no effect (Fig. 4A).
In NOD mice, diabetes is accompanied by infiltration of the
pancreatic islets by autoreactive CTLs. In the 8.3 TCR-transgenic
mice, the vast majority of the islet-infiltrating CD8+T-cell pop-
ulation is specific for H-2Kd/IGRP206–214 (30). These islet-in-
filtrating cells have previously encountered their antigen during
of single-chain tetramers on these in vivo-differentiated CTLs,
islets from diabetic 8.3 TCR-transgenic mice were isolated and
cultured with IL-2 for 6 d. The islet infiltrates that exited the islets
were then treated with scKd.IGRP or scKd.TUM tetramers for 3 h
and analyzed for apoptosis by Annexin V staining. The scKd.IGRP
of these in vivo-differentiated CTLs compared with untreated cells
or those treated with the scKd.TUM tetramers (Fig. 4B).
nate 8.3 T cells. (A) scKd.IGRP monomers (blue line), tetramers (red line), and
molecular weight standards (green dotted line) were analyzed by size-ex-
clusion chromatography on a Superdex 200 column. The standard peaks are
labeled with their molecular weights (in kDa) at the top of the figure. (B)
Splenocytes from 8.3 or AI4 TCR-transgenic NOD mice were stained with anti-
CD8 and the indicated PE-labeled tetramers and analyzed by flow cytometry.
scKd.IGRP tetramers are homogeneous and bind specifically to cog-
| www.pnas.org/cgi/doi/10.1073/pnas.1110971108Samanta et al.
scKd.IGRP Tetramer Treatment Causes Abrogation of Responsiveness
in Islet-Infiltrating Cognate CD8+T Cells. Our studies demonstrated
efficient binding of the scKd.IGRP tetramers to 8.3 T cells (Fig.
2B) and induction of apoptosis in islet-infiltrating CD8+T
cells specific for the IGRP peptide (Fig. 4B). However, there
remained a large number of 8.3 T cells that were Annexin V-
negative. It is of considerable interest to consider the possible
fate of those 8.3 T cells that bound tetramer but did not undergo
apoptosis. A second pathway associated with scKd.IGRP tetra-
mer binding (i.e., TCR engagement in the absence of cos-
timulatory interactions) could be the induction of anergy or
unresponsiveness (7, 8). For this purpose, islet-infiltrating CD8+
T cells from the 8.3 TCR-transgenic mice were treated with the
scKd.IGRP tetramers or the noncognate scKd.TUM tetramers,
and used in an IFN-γ enzyme-linked immunosorbent spot
(ELISPOT) assay to determine their responsiveness. There was
an approximately 80% reduction in the number of spot-forming
cells when the CD8+T cells were treated with the scKd.IGRP
tetramers and presented with the superagonist mimotope pep-
tide NRP-V7 (28), whereas those treated with the scKd.TUM
tetramers retained their responsiveness to the mimotope (Fig. 4C).
Therefore, the binding of the single-chain pMHC tetramers to
cells that have already been activated in vivo, in addition to
causing cell death (Fig. 4B), may also render them nonresponsive
even when presented with a superagonist peptide.
scKd.IGRP Tetramers Can Access Splenic and Islet-Infiltrating T Cells in
Vivo. Given the ability of the scKd.IGRP tetramers to induce ap-
optosis or unresponsiveness of the cognate CD8+T cells, it was of
particular interest to determine whether they could be delivered
in vivo. For these studies, we used tetramers prepared with PE-
labeled streptavidin for the purpose of visualizing them in the
different organs. The 8.3 TCR-transgenic mice were injected i.v.
4 h of treatment, cells from the spleen and the pancreas were
for PE-tetramer binding. CD8+T cells in the spleen and the
pancreas showed tetramer binding only in mice treated with the
scKd.IGRP tetramers (Fig. 5), thus demonstrating the ability of
this reagent to bind to cognate CD8+T cells in vivo. These results
of autoreactivity in type 1 diabetes, and bind the targeted cognate
CD8+T cells. Based on our in vitro results, this delivery would be
expected to ultimately lead to apoptosis and/or the induction of
unresponsiveness in this specific T-cell population.
For naive and antigen-experienced T cells, TCR engagement by
pMHC in the absence of the costimulatory signal provided by
binding of CD28 to its ligands (CD80 and CD86) can result in T-
(A) Splenocytes from 6- to 8-wk-old 8.3 TCR-transgenic NOD mice were in-
cubated at 37 °C for 3 h with 25 nM scKd.IGRP tetramers (red histogram) or
25 nM scKd.TUM tetramers (blue) or left untreated (filled gray), and then
stained with anti-CD8 and anti-CD62L and analyzed by flow cytometry.
Samples were gated on CD8+cells. (B) As in A, except that treated and un-
treated splenocytes were stained with anti-CD8, Annexin V, and 7-AAD and
analyzed by flow cytometry. Samples were gated on CD8+7-AAD−cells. (C)
As in B, except that splenocytes from 6- to 8-wk-old AI4 TCR-transgenic NOD
mice were used. In A–C, numbers denote the percentage of cells present in
the indicated quadrants of the dot plots.
Induction of apoptosis in naive CD8+T cells by scKd.IGRP tetramers.
generated CTLs and modulation of CTL activity by scKd.
IGRP tetramers. (A) In vitro-generated 8.3 CTLs were in-
cubated at 37 °C for 3 h with tetramers of scKd.IGRP or
scKd.TUM at 25 nM. Cells were stained with anti-CD8,
Annexin V, and 7-AAD and analyzed by flow cytometry.
Samples were gated on CD8+7-AAD−cells. (B) As in A,
except that islet-infiltrating cells from 8.3 TCR-transgenic
NOD mice were used. (C) Islet infiltrates from 8.3 TCR-
transgenic mice were incubated at 37 °C for 3 h with tet-
ramers of either scKd.IGRP or scKd.TUM at 25 nM. The T-cell
response to the superagonist peptide NRP-V7 presented by
RMA-S/Kdcells was measured by IFN-γ ELISPOT assay. Spot-
forming cells per 103islet-infiltrating cells are shown.
Induction of apoptosis in in vitro- and in vivo-
Samanta et al.PNAS
| August 16, 2011
| vol. 108
| no. 33
represents the rationale for a variety of therapeutic approaches
that are currently being used or explored for the treatment of
autoimmune disease (32). Although one such effective strategy is
theuseofCTLA4-Ig in thetreatmentofrheumatoid arthritis(33),
antigen-specific approaches might be more desirable, as they
would reduce the increased risk of infections and cancers that can
accompany systemic immunosuppression. Single-chain pMHC
classI molecules were originally designed tobeused inthecontext
of DNA vaccination to augment immune responses, as covalent
linkage of the peptide would result in very stable cell surface
expression of defined tumor- or pathogen-derived peptides (13,
14, 34). Their potential to manipulate autoreactive T cells in
an antigen-specific manner when multimerized is underexplored.
Here we demonstrate that these reagents possess considerable
utility for this purpose, without the potential complication of
peptide transfer to cells with costimulatory properties and the un-
intended activation of naive T cells (11, 12). Such reagents should
pose advantages over both peptide therapy, which suffers from
short peptide half-life in vivo (35) and the risk of anaphylaxis (36),
and administration of antigen-coupled fixed syngeneic cells (37,
38), which will require ex vivo manipulation of a patient’s cells.
The apoptosis, as marked by phosphatidylserine externaliza-
tion, that we observed in naive T cells treated with scKd.IGRP
tetramers is consistent with the requirement for both antigen
exposure and costimulation to support the survival of naive T
cells (9). In contrast, the apoptosis observed upon treatment of
CTLs with the cognate tetramers may be more akin to activation-
induced cell death, such as that observed by others when CD8+
T-cell clones specific for foreign antigens were treated with
multimeric pMHC complexes (29, 39). CD28 costimulation helps
to promote the survival of activated T cells (6), and this cos-
timulatory signal is not provided by pMHC multimers. However,
we observed cell death in only a fraction of the treated autor-
eactive 8.3 CTLs, and the remaining population appeared to
exhibit hyporesponsiveness to antigen, rather than enhanced
effector function. These findings are consistent with the reported
induction of anergy in cognate self-reactive CD4+T cells upon
treatment with class II pMHC dimers (40–43), and suggest that
scKd.IGRP tetramers use multiple mechanisms to interfere with
CTL survival and function.
Investigation of the therapeutic potential of pMHC multimers
in autoimmune disease has focused almost exclusively on the
targeting of CD4+T cells (41–44). A notable exception is the use
of nanoparticles coated with class I pMHC complexes in NOD-
based mouse models of type 1 diabetes (45). Nanoparticle
treatment can delete cognate T cells and bring about the ex-
pansion of low-avidity memory T cells that have autoregulatory
functions, resulting in achievement of both disease prevention
and reversal (45). Class II pMHC dimers have also been shown
to have a beneficial effect, at least in part, by the fostering of
T-cell populations that have regulatory or immunosuppressive
properties (41, 42, 44). As we continue in vivo studies of our
single-chain pMHC tetramers, the activities of induction of ap-
optosis and hyporesponsiveness we observed in vitro will be
evaluated, as will the impact on regulatory T-cell populations.
Although 8.3 TCR-transgenic mice will be used for some of this
future work, even standard NOD mice, in which disease is caused
by a variety of antigen specificities (46), will be amenable to
study, as they have a substantial population of CD8+T cells
reactive to the IGRP206–214peptide (17, 19, 20). Regardless of
the mechanisms at work, we have demonstrated that a single,
readily produced reagent is capable of inducing apoptosis of
naive peptide-specific CD8+T cells and differentiated CTLs,
while at the same time modulating CTL activity. These findings
suggest the potential of such reagents for both early and late
intervention in the course of autoimmune disease progression.
Materials and Methods
Mice. Male 8.3 TCR-transgenic NOD mice (18) were obtained from The
Jackson Laboratory and crossed with NOD mice bred in house to obtain
female 8.3 TCR-transgenic NOD mice for our experiments. AI4 TCR-
transgenic NOD mice (47) were bred in house. All animals were bred and
maintained under specific pathogen-free conditions at the Albert Einstein
College of Medicine in accordance with protocols approved by the in-
stitutional animal care and use committee.
Cloning, Expression, and Purification of Single-Chain pMHC Monomers. The
single-chain constructs of scKd.IGRP and scKd.TUM were expressed and pu-
rified as described in SI Materials and Methods.
Crystallization and Structure Determination. The crystal structure of scKd.IGRP
was solved and analyzed as described in SI Materials and Methods.
Tetramer Preparation. A BirA biotinylation sequence (GLNDIFEAQKIEWHE)
was added to the C terminus of the single-chain construct to generate
streptavidin-mediated tetramers. The protein was expressed and purified as
described earlier and biotinylated by using the BirA enzyme following the
manufacturer’s protocol (Avidity ); free biotin was removed by size-exclusion
chromatography. Tetramers were prepared by adding streptavidin or PE-
labeled streptavidin (BD Biosciences) at a ratio of four molecules of bio-
tinylated scKd.IGRP to one molecule of streptavidin. The formation of tet-
ramers was analyzed by size-exclusion chromatography using a Superdex
200 HR 10/30 prepacked column (Amersham Biosciences). Tetramers of scKd.
TUM were prepared and characterized in an identical fashion.
Flow Cytometry. Flow cytometric studies were performed with a FACSCalibur
or LSR II device (BD Biosciences) and analyzed by using FlowJo software
(Treestar). Labeled monoclonal antibodies to murine CD8α (53-6.7) and
CD62L (MEL-14) were purchased from BD Biosciences.
In Vitro 8.3 CTL Generation. To generate 8.3 CTLs in vitro, splenocytes from 8.3
TCR-transgenic NOD mice were cultured in the presence of mitomycin
C-treated NOD splenocytes and 10 nM NRP-A7 at a ratio of 1:4. After 6 d of
culture, live 8.3 CTLs were purified with Ficoll and used for experiments.
Pancreatic Islet Isolation and Culture of Islet-Infiltrating CTLs. Islets were iso-
lated after perfusion of the pancreas with collagenase P and cultured for 6
d in RPMI medium (RPMI 1640 supplemented with 1 mM sodium pyruvate, 28
μM β-mercaptoethanol, and nonessential amino acids) supplemented with
10% FBS and 50 U/mL recombinant human IL-2 as described previously (48).
Cell Death Assay. Splenocytes or CTLs (1 × 106cells/mL) were resuspended in
RPMI medium containing 10% FBS and incubated in 100-μL aliquots at 37 °C
for 3 h with 25 nM tetramers of scKd.IGRP or scKd.TUM or left untreated.
Cells were washed and stained with FITC-labeled Annexin V and 7-amino-
actinomycin D (7-AAD) according to the manufacturer’s protocol (BD Bio-
sciences) and analyzed by flow cytometry. Dead (i.e., 7-AAD–positive) cells
were excluded from analysis.
8.3 TCR-transgenic NOD mice were injected i.v. with PE-labeled tetramers of
scKd.IGRP or scKd.TUM. After 4 h, cell suspensions of the spleen and pancreas
were stained with anti-CD8 and analyzed by flow cytometry for PE-tetramer
binding. Numbers denote the percentage of cells present in the indicated
quadrants of the dot plots.
scKd.IGRP tetramers access splenic and islet-infiltrating T cells in vivo.
| www.pnas.org/cgi/doi/10.1073/pnas.1110971108Samanta et al.
after 6 d of culture, resuspended in RPMI medium containing 10% FBS, and
incubated in 100-μL aliquots at 37 °C for 3 h with 25 nM tetramers of scKd.IGRP
or scKd.TUM or left untreated. ELISPOT plates (MAHA S45 10; Millipore) were
precoated with anti-mouse IFN-γ mAb R4-6A2 (BD Pharmingen) and blocked
with 1% BSA (Fraction V; Sigma-Aldrich). RMA-S cells engineered to express
the MHC class I molecule H-2Kd(RMA-S/Kd; originally obtained as a gift from
M. Bevan, University of Washington, Seattle, WA) were plated at a density of
2 × 104cells per well and pulsed with 1 μM NRP-V7 peptide for 1 h at 26 °C.
Islet-infiltrating T cells were cocultured with the peptide-pulsed antigen-
presenting cells at 103cells per well for 40 h at 37 °C. IFN-γ secretion was
detected with a second, biotinylated anti-mouse IFN-γ mAb XMG1.2 (BD
Pharmingen) and spots were developed by using streptavidin–alkaline phos-
phatase (Zymed Laboratories) and 5-bromo-4-chloro-3-indolyl-phosphate/
nitroblue tetrazolium substrate (Sigma-Aldrich). Spots were counted using an
automated ELISPOT reader system (Autoimmun Diagnostika).
In Vivo Delivery of scKd.IGRP Tetramers. Tetramers of scKd.IGRP or scKd.TUM
were prepared with PE-labeled streptavidin for the purpose of visualiza-
tion by flow cytometry after in vivo administration. The 8.3 TCR-transgenic
NOD mice were injected i.v. with 200 μL of a 1-μM solution of PE-labeled
tetramers of scKd.IGRP or scKd.TUM. After 4 h, single-cell suspensions of
spleen and pancreas were prepared. To do this, spleens were gently
ground between frosted glass slides. Pancreata were cut into small pieces
in the presence of a protease inhibitor mixture (Sigma-Aldrich) and
digested in RPMI medium containing 5% FBS, 1 mg/mL collagenase IV
(Sigma-Aldrich), 2 U/mL DNase I (Roche), and 1.5 U/mL heparin (Sigma-
Aldrich) for 10 min at 37°C. Digested pancreatic tissue was then passed
through a 40-μm cell strainer. Single-cell suspensions of spleen and pan-
creas were stained with anti-CD8 and analyzed by flow cytometry for PE–
ACKNOWLEDGMENTS. We thank the staff of the X29A beam line at the
National Synchrotron Light Source; Hui Ziao and Wendy Zencheck for help
with MS; and Jeffrey Babad, Eszter Lazar-Molnar, and Laura Santambrogio
for their critical reading of the manuscript. This work was supported by
National Institutes of Health (NIH) Grants AI007289 (to S.G.N. and S.C.A.),
DK064315 (to T.P.D.), and DK020541 (to Albert Einstein College of Medi-
cine’s Diabetes Research and Training Center). The flow cytometry facility at
Albert Einstein College of Medicine is supported by NIH Cancer Center
1. Walter U, Santamaria P (2005) CD8+T cells in autoimmunity. Curr Opin Immunol 17:
2. Angelov GS, et al. (2006) Soluble MHC-peptide complexes containing long rigid
linkers abolish CTL-mediated cytotoxicity. J Immunol 176:3356–3365.
3. Cebecauer M, et al. (2005) Soluble MHC-peptide complexes induce rapid death of
CD8+CTL. J Immunol 174:6809–6819.
4. Dal Porto J, et al. (1993) A soluble divalent class I major histocompatibility complex
molecule inhibits alloreactive T cells at nanomolar concentrations. Proc Natl Acad Sci
5. O’Herrin SM, et al. (2001) Antigen-specific blockade of T cells in vivo using dimeric
MHC peptide. J Immunol 167:2555–2560.
6. Boise LH, et al. (1995) CD28 costimulation can promote T cell survival by enhancing
the expression of Bcl-XL. Immunity 3:87–98.
7. Gimmi CD, Freeman GJ, Gribben JG, Gray G, Nadler LM (1993) Human T-cell clonal
anergy is induced by antigen presentation in the absence of B7 costimulation. Proc
Natl Acad Sci USA 90:6586–6590.
8. Harding FA, McArthur JG, Gross JA, Raulet DH, Allison JP (1992) CD28-mediated sig-
nalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones.
9. Van Parijs L, Ibraghimov A, Abbas AK (1996) The roles of costimulation and Fas in T
cell apoptosis and peripheral tolerance. Immunity 4:321–328.
10. Wang B, Maile R, Greenwood R, Collins EJ, Frelinger JA (2000) Naive CD8+T cells do
not require costimulation for proliferation and differentiation into cytotoxic effector
cells. J Immunol 164:1216–1222.
11. Ge Q, et al. (2002) Soluble peptide-MHC monomers cause activation of CD8+T cells
through transfer of the peptide to T cell MHC molecules. Proc Natl Acad Sci USA 99:
12. Schott E, Bertho N, Ge Q, Maurice MM, Ploegh HL (2002) Class I negative CD8 T cells
reveal the confounding role of peptide-transfer onto CD8 T cells stimulated with
soluble H2-Kbmolecules. Proc Natl Acad Sci USA 99:13735–13740.
13. Mitaksov V, et al. (2007) Structural engineering of pMHC reagents for T cell vaccines
and diagnostics. Chem Biol 14:909–922.
14. Yu YY, Netuschil N, Lybarger L, Connolly JM, Hansen TH (2002) Cutting edge: single-
chain trimers of MHC class I molecules form stable structures that potently stimulate
antigen-specific T cells and B cells. J Immunol 168:3145–3149.
15. Nagata M, Santamaria P, Kawamura T, Utsugi T, Yoon JW (1994) Evidence for the role
of CD8+cytotoxic T cells in the destruction of pancreatic β-cells in nonobese diabetic
mice. J Immunol 152:2042–2050.
16. DiLorenzo TP, Serreze DV (2005) The good turned ugly: immunopathogenic basis for
diabetogenic CD8+T cells in NOD mice. Immunol Rev 204:250–263.
17. Lieberman SM, et al. (2003) Identification of the β cell antigen targeted by a prevalent
population of pathogenic CD8+T cells in autoimmune diabetes. Proc Natl Acad Sci
18. Verdaguer J, et al. (1997) Spontaneous autoimmune diabetes in monoclonal T cell
nonobese diabetic mice. J Exp Med 186:1663–1676.
19. Lieberman SM, et al. (2004) Individual nonobese diabetic mice exhibit unique pat-
terns of CD8+T cell reactivity to three islet antigens, including the newly identified
widely expressed dystrophia myotonica kinase. J Immunol 173:6727–6734.
20. Trudeau JD, et al. (2003) Prediction of spontaneous autoimmune diabetes in NOD
mice by quantification of autoreactive T cells in peripheral blood. J Clin Invest 111:
21. Jarchum I, Nichol L, Trucco M, Santamaria P, DiLorenzo TP (2008) Identification of
novel IGRP epitopes targeted in type 1 diabetes patients. Clin Immunol 127:359–365.
22. Mallone R, et al. (2007) CD8+T-cell responses identify β-cell autoimmunity in human
type 1 diabetes. Diabetes 56:613–621.
23. Standifer NE, et al. (2006) Identification of Novel HLA-A*0201-restricted epitopes in
recent-onset type 1 diabetic subjects and antibody-positive relatives. Diabetes 55:
24. Wallny HJ, et al. (1992) Identification and quantification of a naturally presented
peptide as recognized by cytotoxic T lymphocytes specific for an immunogenic tumor
variant. Int Immunol 4:1085–1090.
25. Mitaksov V, Fremont DH (2006) Structural definition of the H-2Kdpeptide-binding
motif. J Biol Chem 281:10618–10625.
26. Wilson IA, Fremont DH (1993) Structural analysis of MHC class I molecules with bound
peptide antigens. Semin Immunol 5:75–80.
27. Anderson B, Park BJ, Verdaguer J, Amrani A, Santamaria P (1999) Prevalent CD8+T
cell response against one peptide/MHC complex in autoimmune diabetes. Proc Natl
Acad Sci USA 96:9311–9316.
28. Amrani A, et al. (2001) Expansion of the antigenic repertoire of a single T cell receptor
upon T cell activation. J Immunol 167:655–666.
29. Guillaume P, et al. (2003) Soluble major histocompatibility complex-peptide octamers
with impaired CD8 binding selectively induce Fas-dependent apoptosis. J Biol Chem
30. Krishnamurthy B, et al. (2008) Autoimmunity to both proinsulin and IGRP is required
for diabetes in nonobese diabetic 8.3 TCR transgenic mice. J Immunol 180:4458–4464.
31. Zhang Y, et al. (2002) In situ β cell death promotes priming of diabetogenic CD8 T
lymphocytes. J Immunol 168:1466–1472.
32. Podojil JR, Miller SD (2009) Molecular mechanisms of T-cell receptor and cos-
timulatory molecule ligation/blockade in autoimmune disease therapy. Immunol Rev
33. Schiff M (2011) Abatacept treatment for rheumatoid arthritis. Rheumatology (Ox-
34. Hansen TH, Connolly JM, Gould KG, Fremont DH (2010) Basic and translational ap-
plications of engineered MHC class I proteins. Trends Immunol 31:363–369.
35. Ishioka GY, et al. (1994) Failure to demonstrate long-lived MHC saturation both
in vitro and in vivo. Implications for therapeutic potential of MHC-blocking peptides.
J Immunol 152:4310–4319.
36. Liu E, et al. (2002) Anti-peptide autoantibodies and fatal anaphylaxis in NOD mice in
response to insulin self-peptides B:9-23 and B:13-23. J Clin Invest 110:1021–1027.
37. Fife BT, et al. (2006) Insulin-induced remission in new-onset NOD mice is maintained
by the PD-1-PD-L1 pathway. J Exp Med 203:2737–2747.
38. Niens M, et al. (2011) Prevention of “humanized” diabetogenic CD8 T-cell responses
in HLA-transgenic NOD mice by a multipeptide coupled-cell approach. Diabetes 60:
39. Xu XN, et al. (2001) A novel approach to antigen-specific deletion of CTL with minimal
cellular activation using α3 domain mutants of MHC class I/peptide complex. Immu-
40. Appel H, Seth NP, Gauthier L, Wucherpfennig KW (2001) Anergy induction by dimeric
TCR ligands. J Immunol 166:5279–5285.
41. Casares S, et al. (2002) Down-regulation of diabetogenic CD4+T cells by a soluble
dimeric peptide-MHC class II chimera. Nat Immunol 3:383–391.
42. Masteller EL, et al. (2003) Peptide-MHC class II dimers as therapeutics to modulate
antigen-specific T cell responses in autoimmune diabetes. J Immunol 171:5587–5595.
43. Zuo L, et al. (2002) A single-chain class II MHC-IgG3 fusion protein inhibits autoim-
mune arthritis by induction of antigen-specific hyporesponsiveness. J Immunol 168:
44. Li L, Yi Z, Wang B, Tisch R (2009) Suppression of ongoing T cell-mediated autoim-
munity by peptide-MHC class II dimer vaccination. J Immunol 183:4809–4816.
45. Tsai S, et al. (2010) Reversal of autoimmunity by boosting memory-like autor-
egulatory T cells. Immunity 32:568–580.
46. Chaparro RJ, DiLorenzo TP (2010) An update on the use of NOD mice to study au-
toimmune (Type 1) diabetes. Expert Rev Clin Immunol 6:939–955.
47. Graser RT, et al. (2000) Identification of a CD8 T cell that can independently mediate
autoimmune diabetes development in the complete absence of CD4 T cell helper
functions. J Immunol 164:3913–3918.
48. Jarchum I, Takaki T, DiLorenzo TP (2008) Efficient culture of CD8+T cells from the
islets of NOD mice and their use for the study of autoreactive specificities. J Immunol
Samanta et al. PNAS
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| vol. 108
| no. 33