Control of antigen presentation with a photoreleasable agonist peptide

Article abstract of DOI:10.1021/ja065304l

The immunological synapse is a specialized intercellular junction between a T cell and a target cell that orchestrates the engagement of receptors and ligands in space and time as a means of regulating function. Here we introduce a reagent for controlling the spatial and temporal presentation of natural antigen to T cells. Moth cytochrome c (88-103) peptide (MCC), an agonist to the murine T cell receptor AND when presented in the context of H2 IE^k major histocompatibility complex (IE^k), was synthesized with the side-chain amine of Lys99 conjugated to a photosensitive protecting group, 6-nitroveratryloxycarbonyl (NVOC). Cells plated on supported bilayers displaying mobile intercellular adhesion molecule-1 (ICAM-1) and NVOC-MCC loaded IEk did not form immunological synapses and exhibited low intracellular calcium levels, similar to cells presented with self-peptide. Irradiation with UV light was sufficient to restore agonist activity in situ. Copyright

Full text of DOI:10.1021/ja065304l

Published on Web 11/10/2006
Control of Antigen Presentation with a Photoreleasable Agonist Peptide
Andrew L. DeMond,†,‡ Toby Starr, Michael L. Dustin, and Jay T. Groves*
§
§
,†,‡
Biophysics Graduate Group and Department of Chemistry, UniVersity of California, Berkeley, California 94720 and
Department of Pathology, Skirball Institute, New York UniVersity School of Medicine, New York, New York 10016
Received July 24, 2006; E-mail: jtgroves@lbl.gov
T cells survey target cells by scanning for antigenic peptides
presented by major histocompatibility complex molecules (pMHC)
1
with T cell receptor (TCR). Engagement of pMHC by TCR induces
a cascade of signaling events, including a rise in intracellular
calcium levels and the formation of a large scale spatial pattern of
signaling and adhesion molecules called the immunological synapse
3
(
IS). Recent studies suggest a direct role for the spatial pattern of
molecules in the synapse with respect to regulation of TCR
signaling.4 The interaction between TCR and pMHC is sensitive
to point mutations in the antigenic peptide; mutated agonist peptides
induce a panoply of responses in the T cell ranging from attenuated
-6
7
activation to antagonism to null. In the well studied TCR and
k
pMHC pair of AND and MCC-IE (moth cytochrome c, residues
8
8-103),8 replacing the critical lysine at position 99 with
,9
glutamate (and thus flipping the charge) diminishes the agonist
effect of the peptide. On the basis of the crystal structure of the
MCC-IEk and its putative interaction with AND TCR,2 we
hypothesized that the presence of a bulky caging group on the end
of the critical K99 residue of MCC would abolish activation (Figure
Figure 1. Experimental setup: (Ai) T cell receptor (TCR) weakly binds
k
moth cytochrome c (88-103) (MCC) peptide presented by IE major
k
histocompatibility complex (IE ) when the peptide is derivatized on Lys99
with the light sensitive protecting group 6-nitroveratryloxycarbonyl (NVOC).
(
Aii) Near-UV light cleaves the protecting group, restoring (Aiii) strong
1). Caged proteins and peptides are powerful tools for controlling
k
binding between TCR and MCC-IE . (B) The crystal structure of MCC-
IE
cellular signaling. 6-Nitroveratryloxycarbonyl (NVOC) was orig-
k,2
shown in ribbon form, underscores the importance of Lys99,
inally introduced as a protecting group for solid-phase peptide
synthesis10 and has since been employed in a variety of spatially
highlighted against the white space-filling MCC peptide. (C) A cartoon of
NVOC derivatized MCC peptide illustrates NVOC mediated steric hindrance
of TCR binding to MCC-IE .
k
and temporally modulated chemical reactions, in particular involving
amino acids and peptides.11,12 It can be readily linked to terminal
amines and is cleaved by near-UV light. Here we show that caged
antigenic peptide can be used to selectively activate T cells in a
spatially and temporally controlled fashion. This strategy provides
a straightforward way to pattern two-dimensional arrangements of
molecules for probing the spatial regulation of T-cell signaling.
A peptide variant of MCC in which the Lys99 residue critical
to MHC-TCR interaction was derivatized with NVOC was
synthesized using solid-phase peptide synthesis (Figure S1).13 The
UV-light-induced uncaging rate of the peptide was determined by
mass spectrometry (Supporting Information, Figure S2). All experi-
ments were performed in the presence of 50 µM dithiothreitol
Because of the uncaged peptide contamination, it was necessary to
dilute the MCC or NVOC-MCC in a background of the non-
activating peptide MCC-T102E, in which the threonine at position
3
1
02 has been replaced by glutamate. The surface density ratio of
k
activating to caged MCC-IE could not be determined directly;
assuming that caging K99 does not modify the binding constant of
k
MCC to IE , a 1:25 dilution of MCC/MCC-T102E with a surface
k
2
density of 50 IE /µm and a loading efficiency of ∼50% yields an
2
approximate surface density of 1-2 caged peptides/µm , which is
3
above the activating threshold for MCC on bilayers. The amount
of uncaged MCC impurity in NVOC-MCC experiments was
2
determined to be approximately 0.02/µm , which is subthreshold
(
DTT) to decrease secondary reactions of the nitrosoacetophenone
(B. Rossenova, personal communication). At this very low density
14
photoproduct which can chemically cross-link proteins. The
addition of 50 µM DTT did not interfere with protein stability.
The basal level of uncaged MCC contamination due to sample
manipulation in dimmed room light was determined to be ∼1%.
Uncaging was also confirmed with the TCR-mimic antibody D4
of NVOC-MCC, at most 200 molecules of the photoproduct
nitrosoacetophenone are generated in the vicinity of a cell during
uncaging. Any molecules not immediately quenched by the DTT
in solution would nonspecifically interact with cell surface mol-
ecules, and thus it is unlikely that the photoproducts produce any
cellular effect.
Intracellular calcium flux, which occurs downstream of TCR
binding in T-cell activation, was examined in cells stimulated with
agonist, caged and self-peptide. T-cell blasts were loaded with the
calcium-sensitive dye Fluo-Lojo and then plated onto peptide-
displaying bilayers. Cells adopted a rounded morphology and fluxed
calcium when stimulated with MCC bilayers (Figure 2). On
NVOC-MCC bilayers, cells crawled, showing a low level of
intracellular calcium similar to cells on T102E bilayers. NVOC-
(Figure S3).
The functionality of NVOC-MCC with live T cells was
examined. T-cell blasts were plated on planar bilayers containing
mobile intercellular adhesion molecule-1 (ICAM) and peptide-
k
loaded IE . Planar bilayers are well established in the study of the
immunological synapse and are stable over the course of days.3
†
Biophysics Graduate Group, University of California.
‡
Department of Chemistry, University of California.
§
New York University School of Medicine.
15354
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J. AM. CHEM. SOC. 2006, 128, 15354-15355
10.1021/ja065304l CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Control of T-cell signaling with caged peptide. (A) Cells plated
k
on bilayers displaying IE and ICAM-1 loaded with normal MCC form the
classic immunological synapse, with a central accumulation of TCR
surrounded by a peripheral ring of ICAM-1. Cells stimulated with caged
NVOC-MCC peptide display diffuse TCR and ICAM. (B) A migrating
cell displaying the stereotypical hand-mirror shape and ICAM wedge was
2
irradiated with UV (80 nW/µm , 60 s). Upon UV exposure, the cell rounded
up and formed the classic ICAM ring, indicative of synaptic signaling (signal
onset was within 5 min). (C) On bilayers displaying only the null peptide
MCC-T102E, UV irradiation does not cause any signaling. At 0 min, a
2
crawling cell was irradiated with UV (80 nW/µm , 30 s). Just after
irradiation, the cell retained the hand-mirror morphology. At 15 min, the
cell continues to crawl on the bilayer, indicating no damage or signaling.
Scalebars are 10 µm.
induce activation. These T cells were probably in an internal state
unreceptive to activation, possibly owing to mild antagonism by
MCC-T102E.
Figure 2. UV light restores agonist activity of caged peptide. (A) Cells
loaded with the calcium-sensitive dye Fluo-Lojo strongly flux calcium when
stimulated by bilayers displaying the agonist peptide MCC diluted in a
background of the self-peptide T102E. (B) When plated on bilayers
displaying caged NVOC-MCC peptide in the same dilution, cells do not
flux calcium and adopt a crawling morphology, similar to the response (C)
of cells to bilayers displaying only the self-peptide T102E. (D) Pre-
irradiation of NVOC-MCC loaded bilayers with UV light (15 min, 0.5
NVOC-MCC is an effective reagent for specific activation of
T-cell signaling on supported bilayers. Using a standard epi-
fluorescence microscope, T-cell signaling and immune synapse
formation can be activated in a spatially and temporally controlled
fashion. Future applications of photoreleasable peptide are most
promising in the area of subcellular protein patterning. By com-
bining nanofabricated barriers to diffusion with photolithography
of activating peptide, study of the effects of subcellular juxtaposition
of agonist and nonactivating peptide signals on T-cell activation
will be directly accessible.
2
nW/µm , 97% uncaged) rescues the stimulatory effect of the caged peptide.
Cell adhesion was confirmed with reflection interference contrast micros-
copy; dark regions indicate close adherence to the bilayer. Scalebar is 10
µm.
MCC bilayers exposed to UV light before the addition of cells
behaved similarly to MCC bilayers. Extended illumination with
>450 nm light did not uncage the NVOC, indicating visible light
fluorescence imaging is compatible with the caged peptide. These
data suggest that UV light successfully exposes the original MCC
peptide from NVOC-MCC in situ.
Acknowledgment. We thank D. King of the HHMI Mass
Spectrometry Facility for peptide synthesis and mass spectrometry.
N. Switz, M. B. Forstner, A. Liu, and K. Mossman provided
valuable discussion. A.L.D. is supported by an NSF Graduate
Research Fellowship. M.L.D. is supported by NIH grant AI044931.
Caged peptide also affects immune synapse formation. Cells that
were stimulated with bilayers loaded with 1:25 MCC/T102E
strongly tended to stop and form the characteristic immune synapse
pattern of a strong central accumulation of TCR surrounded by a
ring of ICAM (Figure 3A). In contrast, cells stimulated with bilayers
displaying NVOC-MCC did not form synapses. TCR was diffuse
over the face of these cells, whereas activated cells displayed the
classic central accumulation of TCR. The aggregate behavior is
Supporting Information Available: Experimental procedures and
supplementary figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
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