High-resolution, noninvasive longitudinal live imaging of immune responses

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16 Νοε 2013 (πριν από 3 χρόνια και 11 μήνες)

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High-resolution,noninvasive longitudinal live imaging
of immune responses
Midhat H.Abdulreda
a,1
,Gaetano Faleo
a,1
,Ruth Damaris Molano
a
,Maite Lopez-Cabezas
a
,Judith Molina
a,b
,
Yaohong Tan
c
,Oscar A.Ron Echeverria
a
,Elsie Zahr-Akrawi
a
,Rayner Rodriguez-Diaz
a
,Patrick K.Edlund
d
,Ingo Leibiger
d
,
Allison L.Bayer
a,e
,Victor Perez
c,e
,Camillo Ricordi
a,b,d,e,f,g
,Alejandro Caicedo
a,b,f,h,2,3
,Antonello Pileggi
a,e,f,g,2,3
,
and Per-Olof Berggren
a,d,2,3
a
Diabetes Research Institute and
g
Department of Biomedical Engineering,University of Miami,Miami,FL 33136;Departments of
b
Medicine,
e
Microbiology
and Immunology,and
h
Physiology and Biophysics;
c
Bascom Palmer Eye Institute,and
f
DeWitt-Daughtry Department of Surgery,Division of Cellular
Transplantation,University of Miami Miller School of Medicine,Miami,FL 33136;and
d
The Rolf Luft Research Center for Diabetes and Endocrinology,
Karolinska Institutet,Stockholm SE-171 76,Sweden
Edited* by Michael D.Cahalan,University of California,Irvine,CA,and approved June 15,2011 (received for review March 30,2011)
Intravital imaging emerged as an indispensible tool in biological
research,and a variety of imaging techniques have been de-
veloped to noninvasively monitor tissues in vivo.However,most
of the current techniques lack the resolution to study events at the
single-cell level.Although intravital multiphoton microscopy has
addressed this limitation,the need for repeated noninvasive
access to the same tissue in longitudinal in vivo studies remains
largely unmet.We now report on a previously unexplored ap-
proach to study immune responses after transplantation of pan-
creatic islets into the anterior chamber of the mouse eye.This
approach enabled (i) longitudinal,noninvasive imaging of trans-
planted tissues in vivo;(ii) in vivo cytolabeling to assess cellular
phenotype and viability in situ;(iii) local intervention by topical
application or intraocular injection;and (iv) real-time tracking of
infiltrating immune cells in the target tissue.
allorejection
|
in vivo imaging
|
T-cell dynamics
|
infiltration
|
islet grafts
R
ecent advances in intravital microscopy have enabled visual-
ization and quantification of key biological processes in the
physiological context of the natural environment in situ (1),re-
vealing phenomena not predicted by in vitro studies.This insight
has spurred a need for intravital imaging approaches that enable
combined noninvasive and longitudinal monitoring of the same
target tissue with cellular resolution.Techniques such as magnetic
resonance imaging and positron emission tomography or bio-
luminescence (2) have enabled noninvasive visualization of
organs/tissues deep within the body by relying on macroscopic
and indirect parameters (3,4).However,even with the use of
high-contrast materials or tissue-specific luminescence,these
techniques cannot achieve single-cell level sensitivity because of
high background signals and low spatial resolution (5).These
limitations were addressed with the advent of multiphoton mi-
croscopy (6),that enabled high-resolution intravital imaging.
Recently,intravital immunoimaging studies adopting multiphoton
microscopy revealed complex dynamic behaviors of immune cells
(1) crucial for immune function.Most studies,however,have
mainly focused on the immune cell behavior during the priming
phase in lymph nodes (7–9),and a few studies have addressed the
movement of T-effector (T
eff
) lymphocytes within solid tumors or
autoimmune encephalomyelitis lesions (10–12).Very recently,
the kinetics of dendritic cells and T
eff
cells during the phases of
cutaneous graft rejection have been described using multiphoton
microscopy (13).Therefore,availability of new analytical tools
with increased resolution is furthering the horizons of immu-
nobiology,with the characterization of novel biological phe-
nomena associated with 3D dynamic behavior of immune cells
within target tissues during their destruction in vivo,that is im-
probable to predict based in ex vivo and in vitro assays.
Pancreatic islets have been extensively used in animal models
to study immune processes (14).Islet cells are subject to immune
attack during autoimmunity (i.e.,type 1 diabetes) and during
allorejection after transplantation,making them an ideal exper-
imental model with clinical relevance.In small-animal models,
pancreatic islets are commonly transplanted under the kidney
capsule.This process has allowed reliable monitoring of graft
function and survival noninvasively based on metabolic readouts
(e.g.,glycemia).However,in vivo imaging of subcapsular islet
grafts is cumbersome,as it requires surgical exposure of the
kidney precluding the ability to image longitudinally the same
islets (15).Additionally,the imaging resolution is severely
compromised by the thick kidney capsule.The need for invasive
surgical access to the pancreas also limits the number of re-
peated sessions to image islet autoimmunity (1,16).We have
previously demonstrated that individual syngeneic pancreatic
islets transplanted into the anterior chamber of the mouse eye
can be repeatedly imaged with single-cell resolution (17,18).
Here,we describe that the intraocular islet transplantation
model is well suited to study immune cell responses by live mi-
croscopy.This model thus enables high-resolution longitudinal
analysis in the same tissue of the dynamic patterns and inter-
actions of immune cells with target cells,and allows in situ
cytolabeling and acute modulation of the transplant microenvi-
ronment in the living animal.
Results
Longitudinal Imaging of T
eff
Cell Infiltration and Dynamics in Target
Tissues in Vivo.
To study the in vivo kinetics and dynamics of
cellular movement and interactions during rejection of alloge-
neic tissues,we used a model of fully MHC-mismatched pan-
creatic islet transplantation without immunosuppression to
evaluate the natural history of the immune response.DBA/2
(H-2
d
) mouse islets were implanted into the anterior chamber of
the eye of immune competent C57BL/6 (H-2
b
) recipients that
express GFP in activated and memory T lymphocytes (B6.129P2-
Cxcr6tm1Litt/J) (19) (Fig.1 A and B).Similarly to what has been
Author contributions:M.H.A.,R.D.M.,V.P.,C.R.,A.C.,A.P.,and P.-O.B.designed research;
M.H.A.,G.F.,M.L.-C.,J.M.,Y.T.,E.Z.-A.,R.R.-D.,and A.C.performed research;M.H.A.,
R.D.M.,P.K.E.,I.L.,A.L.B.,and A.P.contributed new reagents/analytic tools;M.H.A.,
G.F.,and O.A.R.E.analyzed data;and M.H.A.,G.F.,R.D.M.,A.C.,A.P.,and P.-O.B.wrote
the paper.
Conflict of interest statement:P.-O.B.is one of the founders of the biotech company
Biocrine,which is going to use the anterior chamber of the eye as a commercial servicing
platform.A.C.and I.L.are also involved in the commercialization of this servicing
platform.
*This Direct Submission article had a prearranged editor.
1
M.H.A.and G.F.contributed equally to this work.
2
A.C.,A.P.,and P.-O.B.contributed equally to this work.
3
To whomcorrespondence may be addressed.E-mail:acaicedo@med.miami.edu,apileggi@
med.miami.edu,or per-olof.berggren@ki.se.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1105002108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1105002108 PNAS Early Edition
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MEDICALSCIENCES
reported in islets transplanted under the kidney capsule (15),our
longitudinal noninvasive in vivo imaging studies in the same islet
grafts revealed that the GFP-labeled T-cell infiltrate increased
substantially around and within the allografts at postoperative
day (POD) +14,and that T-cell infiltration paralleled islet de-
struction (Fig.1C and Fig.S1).Furthermore,when adequate
numbers of allogeneic islets were transplanted into chemically
induced diabetic animals,allograft rejection invariably resulted
in loss of graft function (Fig.1D).No infiltration was seen in
control syngeneic islets which were intact during the follow up
and never lost function (Fig.1 A–D).These results demonstrate
that allorejection of pancreatic islets occurs in the anterior
chamber of the eye despite the notion of being an immune
privileged site (20,21).This finding was further confirmed by
typical priming and lymphocyte activation in associated lymph
nodes (axillary and cervical) and spleens of intraocular allograft
recipients (Fig.1E),even though rejection of intraocular islet
grafts appeared to be delayed compared with those under the
kidney capsule.
We next examined the movement of infiltrating GFP-labeled
T lymphocytes in 3D time-lapse recordings lasting 20 min (4D
imaging) (Fig.1 F and G,and Movie S1).The recordings were
acquired in the same islet grafts at different time points non-
invasively (Movie S2).Because islet rejection was not a syn-
chronous phenomenon,we used the day on which the volume of
individual islets was reduced by ≥30% as rejection onset (re-
jection time 0 ± 1 d),which we termed “acute phase” of re-
jection.Quantitative tracking of GFP-labeled T cells within the
islets revealed significant changes in T-cell movement during
acute rejection compared with ≥3 d before rejection (Fig.1H
and Fig.S1F).Taken together,these results demonstrate that
intraocular transplantation enables longitudinal in vivo imaging
Fig.1.Transplantation into the anterior chamber (ACE) of the mouse eye enables longitudinal,noninvasive imaging and tracking of immune cells in in-
dividual islet grafts in vivo.(A) Images of mouse eyes transplanted with allogeneic or syngeneic islets that have engrafted on the iris.Allogeneic islets dis-
appeared by day 14 after transplantation because of rejection.(B) In vivo confocal z-stacks (shown as maximum projections) of the same intraocular grafts
showing progressive infiltration of allogeneic grafts by GFP-labeled (green) T lymphocytes.(Scale bar,100 μm.) (C) Change in the average islet volume (circles)
vs.the number (squares) of graft-infiltrating T-cells (blue,allogeneic;red,syngeneic).Data based on 5 to 31 islets/time point from 5 allogeneic,and 5 to 22
islets/time point fromthree syngeneic recipients.Results presented as means ± SEM.(D) Survival curves of islet grafts in the ACE or under the kidney capsule
(KDN) based on glycemia (gly) or volume (vol) [Syn ACE:n = 4;Allo ACE (gly):n = 7;Allo ACE (vol):n = 12;Allo KDN:n = 24].(E) IL-2 and IFN-γ production
(ELISPOT) in response to alloantigen challenge in cervical/axillary lymph nodes (n = 3 mice) and spleens (n = 3) of nontransplanted (no TX) or transplanted (TX)
animals in the ACE.(F) Snapshot (XYZ view) froma 3D time-lapse recording (20 min;POD +21) showing a tracked cell within an islet allograft (outlined with
dotted line).(Scale bars,10 μm.).(G) Two-dimensional flower plot representation of movement trajectories of individual cells tracked (in 3D) within the graft.
(H) Dynamic parameters derived from 3D tracking analysis on infiltrating GFP-labeled T cells.Velocity (average speed/path length) and meandering index
(displacement rate/velocity;a measure of movement directionality) were measured during 20-min recordings obtained in the same islet allografts at
the indicated time points (± 1 d).Data derived from> 2,000 graft-infiltrating cells and pooled from23 islets in seven mice.Results presented as means ± SEM
(n = 3–6 islets per time point).*P < 0.05.
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with single-cell resolution of immunological events within target
tissues noninvasively.
Fluorescence Labeling of Target and Immune Cells in Vivo.
We
injected fluorescence-labeled antibodies directly into the ante-
rior chamber of the eye at different time points after trans-
plantation to assess the phenotype of the graft-infiltrating
GFP-labeled immune cells in vivo (22).The majority of the an-
tibody-labeled GFP
+
T cells within allogeneic grafts was CD8
+
(≥80%) and few were CD4
+
(<10%) (Fig.2A and Movie S3).
Most GFP-labeled CD8
+
T-cells expressed CD25 and none were
positive for L-selectin (CD62L),confirming their activated status.
These cells also expressed lymphocyte function-associated anti-
gen-1 (LFA-1),which is essential for leukocyte extravasation upon
activation (23) (Fig.S2A).Control isotype antibodies confirmed
specific labeling.Flow cytometry analysis of GFP-labeled lym-
phocytes in lymph nodes derived from intraocular islet allograft
recipients showed similar proportions of CD8
+
and CD4
+
T-cells
(78.2 ± 1.2% and 12.3 ± 1.2%,respectively) (Fig.S2B).
We also labeled intraocular target islet cells with early
(annexin V) and late (DAPI) apoptotic markers.In vivo 4D
imaging during acute rejection showed close association of
“ruffled” GFP-labeled T cells with annexin and DAPI-labeled
islet cells (Fig.2B and Movie S4).Approximately 70% of the
graft-infiltrating GFP-labeled T cells contacted apoptotic islet
cells.Given the focal destruction of the islets near clusters of
GFP-labeled T-cells,we further assessed these cells for effector
function (Fig.2C).In vivo dynamic studies showed the presence
of lytic granules (visualized by intraocular Lysotracker injection)
within GFP-labeled T cells and showed their polarization toward
multiple target islet cells (24) (Movie S5).Examination of frozen
sections of intraocular islet grafts demonstrated a similar staining
pattern of the intracellular cytolytic enzymes granzyme B and
perforin in graft-infiltrating GFP-labeled T cells (Fig.S2),thus
confirming our in vivo findings.
Four-dimensional imaging further allowed discerning different
morphologies and behaviors of graft-infiltrating T lymphocytes.
We consistently identified three phenotypes:round,elongated,
and ruffled cells (Fig.2E and Movie S6).Round cells were sta-
tionary and commonly found surrounding the allografts early
after transplantation,whereas elongated cells traveled long dis-
tances with a mean instantaneous velocity of ∼3.5 μm/min.
Ruffled cells,however,formed clusters within the islets and
exhibited a complex dynamic behavior (Fig.S2 and Movie S7).
Interestingly,both fraction and number of ruffled cells increased
significantly during acute rejection (Fig.2F).Similar clustering of
T cells was also observed under the kidney capsule by invasive
in vivo imaging performed in a single session during acute re-
jection (Fig.2D).These results highlight the utility of our ap-
proach in assessing cellular phenotypes and viability in
longitudinal noninvasive in vivo imaging studies.
Direct Pharmacological Manipulation of T
eff
Cells in Graft Tissue in
Vivo.
Local pharmacological intervention in target tissue is diffi-
cult in vivo.However,ophthalmologists routinely inject sub-
stances into the anterior chamber of the eye for diagnostic or
Fig.2.In situ labeling of islet cells and graft-infiltrating immune cells in the anterior chamber of the eye in vivo.(A) We injected fluorescence-labeled
antibodies directly into the anterior chamber of the eye between POD +8 and POD +23 to reveal the phenotypes of graft-infiltrating immune cells;∼80%of
the graft-infiltrating GFP-labeled T-cells were CD8
+
.(Scale bar,5 μm.) (B) Ruffled T cells (green) associated with apoptotic islet cells (Annexin V,red;DAPI,
blue) during rejection (Movie S4);∼70%of graft-infiltrating GFP-labeled T cells contacted apoptotic islet cells.*P < 0.05.(Scale bar,10 μm.) (C) Endocrine islet
cells (outlined;Left) and lytic granules (arrowheads) within GFP-labeled ruffled T-cells (green) were in vivo labeled with Lysotracker (red) (Movie S5).Lytic
granules within ruffled T-cells faced target islet cells (Center).Immunohistochemistry of fixed intraocular islet allograft showed the presence of granzyme B/
perforin (arrowheads;Right) in a graft-infiltrating ruffled T lymphocyte (green) (Fig.S2C).Shown images are representative of a minimum of triplicate
experiments.(Scale bars,5 μm.) (D) In vivo confocal images (maximum projections) of GFP-labeled T cells within subcapsular pancreatic islet grafts after
exposure of the kidney at onset of rejection (confirmed by glycemia) at POD +12.Although we were not able to image the islets because of the thick kidney
capsule (gray reflection;Left),we were able to recover GFP signal from infiltrating T-cells (green).[Scale bars,10 μm (Right),30 μm (Center),and 100 μm
(Left).] (E) Close-ups of round,elongated,and ruffled cells with corresponding movement tracks.(Scale bar,5 μm.) (F) Number of round,elongated,and
ruffled cells within the same islet allografts at different time points.Results pooled from 22 islets (n = 3–6 islets/time point in six mice).
Abdulreda et al.PNAS Early Edition
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MEDICALSCIENCES
therapeutic purposes without producing systemic effects.We
injected TAK-779,a specific antagonist of the chemokine recep-
tors CCR5 and CXCR3 (25,26),directly into the anterior
chamber of the eye.Within 10 min of TAK-779 treatment,we
observed significant phenotypic and dynamic changes in the in-
dividual graft-infiltrating GFP-labeled T cells (Fig.3A and
Movie S8).We tracked single cells and found that the majority of
the Tcells converted froma highly dynamic to a round,stationary
phenotype after TAK-779 treatment.Subsequent local adminis-
tration in the same eye of CXCL9/CXCL10,the natural ligands to
CXCR3,reversed the effects of TAK-779 and resulted in signifi-
cant phenotypic and dynamic changes (Fig.3B).
We also performed studies using systemic,chronic blockade of
CCR5/CXCR3 with TAK-779.Longitudinal in vivo imaging in
treated animals showed reduced initial infiltration of the
islet allografts by T cells followed by similar numbers during
onset,albeit delayed,acute rejection compared with untreated
animals (Fig.3C).However,the relative proportion of the ruf-
fled cells within the grafts was significantly reduced (Fig.S3A).
This treatment also resulted in significantly slower overall T-cell
velocity (Fig.3D).Importantly,the lower proportion of ruffled
cells and the slower movement dynamics associated with slower
destruction of the allografts (Fig.S3B) and delayed rejection
(Fig.3E).Together,these results show a primary role of ruffled
T
eff
cells in allorejection of pancreatic islets.These results also
demonstrate that our approach has the spatial and temporal
resolution to assess acute and longitudinal effects of interventions
within target tissues in the living organism noninvasively.
Discussion
We report on a unique approach that combines transplantation
into the anterior chamber of the mouse eye and high-resolution
confocal microscopy to enable longitudinal,noninvasive imaging
of immune responses within target tissues during allorejection,
with unprecedented detail in vivo.This approach also enables in
situ fluorescence cytolabeling and local pharmacological in-
tervention by intraocular injection or topical application.To
demonstrate the versatility of our approach,we used pancreatic
islets as an example because they are subject to immune attack in
autoimmune diabetes and during allorejection after trans-
plantation.Typical immune activation was observed in lymphoid
organs and destruction of intraocular islets invariably occurred
despite the putative “immune privilege” properties of the ante-
rior chamber of the eye.This result is possibly due to the in-
flammation (“danger”) generated with the transplant procedure
and to the ability of isolated islets to produce proinflammatory
and proangiogenic factors that lead to revascularization of the
islet grafts (Fig.S1E).
During progression of immune cell-mediated destruction of
islet allografts,we examined the morphology and dynamic be-
havior of T
eff
cells using longitudinal noninvasive in vivo imaging
in the same islet grafts.We found that a ruffled phenotype of
CD8
+
T
eff
cells predominated during acute rejection and was
likely responsible for the significantly increased net translational
movement during this phase.This behavior is likely the result of
the polyclonal reaction to alloantigens and to the lack of specific
antigen recognition in the context of MHC molecules on target
Fig.3.Intraocular transplantation enables longitudinal noninvasive monitoring after local or systemic pharmacological interventions.(A) Fluorescence
confocal images (maximumprojections) shown in intensity scale to illustrate morphological changes of allograft-infiltrating T cells before and after TAK-779
(50 μM) or CXCL9/CXCL10 (100 nM) acute treatment at the time of rejection (POD +22).(Scale bar,40 μm.) Our approach revealed noticeable and reversible
changes in the cellular morphology and behavior of individual T cells after successive injection of either TAK-779 or CXCL9/CXCL10 into the same eye.(B) We
measured significant changes in the velocity and displacement (10 min) of infiltrating T-cells after these treatments.Data derived from 837 (before),335
(TAK-779),and 340 (CXCL9/CXCL10) GFP-labeled cells,obtained fromthree islets (three mice) imaged at POD +13,+16,+21,and +22.(C and D) Systemic TAK-
779 treatment (250 μg/day;intraperitoneally) between POD +10 and POD +17 resulted in significantly reduced islet graft infiltration by T lymphocytes and
reduced overall movement dynamics.Circles represent islet volume and squares the number of intraislet T-cells in TAK-779-treated (filled symbols;n = 15 islets
in three mice) or untreated animals (open symbols;n = 22 islets in five mice).Results presented as means ± SEM.(E) Systemic TAK-779 treatment delayed islet
rejection (median survival time based on islet volume loss of ≥ 30% = 42 vs.21 d).*P < 0.05.
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cells,which is required for the formation of central supramo-
lecular clusters and establishment of segregated secretory im-
munological synapses (27–29).Indeed,our results showed lack of
CD8 and LFA-1 clustering and no segregation of lytic granules
toward contact zones with single targets despite expression of its
ligand intercellular adhesion molecule-1 on the islet cells (30,
31).Although further studies are needed to fully characterize
this dynamic behavior and elaborate on its biological signifi-
cance,it is likely that it favored simultaneous short contacts with
multiple target islet cells,promoting surface exploration and
antigen sampling,and possibly ensuring efficient and timely
target killing during islet allorejection (7,32,33).This theory is
further supported by our findings after blockade of the chemo-
kine receptors CCR5 and CXCR3.
Chemokines and their receptors play an essential role in im-
mune cell trafficking.Using our approach,we acutely induced
changes in the morphology and dynamic behavior of graft-in-
filtrating T
eff
cells by locally modulating the CCR5/CXCR3 sig-
naling pathways.CCR5/CXCR3 and their ligands are up-
regulated during rejection on infiltrating CD8
+
T
eff
cells and in
pancreatic islets,respectively (34–37).Following sequential
treatments with TAK-779 and CXCL9/CXCL10 in the same eye,
individual T cells transitioned between the stationary,round,and
the motile,elongated phenotypes.More importantly,chronic
CCR5/CXCR3 blockade reduced the ruffled phenotype in the
T
eff
cell infiltrate,slowed their movement dynamics,and was
associated with delayed graft rejection.
In summary,our findings demonstrate that transplantation
into the anterior chamber of the eye provides a versatile exper-
imental tool that enables longitudinal,noninvasive in vivo im-
aging of immune responses within target tissues with cellular
resolution.This process allows studying cell–cell interactions
within target tissues and visualizing cell signaling and motility in
situ.Being able to noninvasively monitor the same target tissue
and study immune responses longitudinally provides new ex-
perimental readouts that can be used in transplantation,cancer,
and autoimmune biology.This process also provides a platform
for drug screening where continuous monitoring of target tissue
can be performed noninvasively in the anterior chamber of the
eye after local or systemic intervention in vivo.Future studies
combining our approach and humanized mouse models (38,39)
will improve our understanding of immune cell dynamics in
human disease and may ultimately yield new therapies.
Materials and Methods
Pancreatic Islet Isolation and Transplantation.Animal procedures were ap-
proved by the University of Miami International Animal Care and Use
Committee.Murine pancreatic islets were isolated as previously described
(40).Isolated islets were cultured overnight and transplanted into the an-
terior chamber of the eye or under the kidney capsule,as previously de-
scribed (17,18).
In Vivo Imaging in the Anterior Chamber of the Mouse Eye.As described
previously (17,18),we imaged the same intraocular islet grafts repeatedly at
the indicated time points using 3D single-photon fluorescence confocal mi-
croscopy.In 4D recordings,in addition to the isoflurane anesthesia,mice
were intraperitoneally injected with 50 μL xylazine (5 mg/mL;AnaSed) ∼10
min before imaging.Islet cells were visualized using reflected laser (back-
scatter).Blood vessels were visualized by TRITC-dextran (2 × 10
6
M.W.;
Invitrogen) injected via the tail vein (0.1 mL of 10 mg/mL solution).
Image Analysis.Z-stacks of 512 × 512 pixels (0.1–0.75 μmper pixel) xy sections
with 0.5- to 3-μm z-spacing were acquired using the resonant scanner,built
into a Leica SP5 imaging system(17).Z-stack images were denoised and con-
trast-enhancedusingVolocity software (Improvision;PerkinElmer).The z-stack
thickness was adjusted to span the whole imaged islet.Analysis of islet volume
and T-cell counts were performed semiautomatically with Volocity.The islet
volume was measured based on a 3D region of interest outlining the whole
islet.The T cells inside the region of interest (i.e.,islet) were automatically
counted by the software based on the GFP fluorescence.In 4D recordings,
z-stacks were acquiredevery 1 to3 minfor 20 min.Time-lapseregistration(i.e.,
drift correction) was done manually with Volocity using static reference points
inthereflectionchannel (namely,islets).Movies wereautomatically generated
fromregistered 4Drecordings.We tracked >2,000 T lymphocytes inregistered
time-lapse studies acquired at different time points on the same allogeneic
islets.All dynamic parameters were derived based on 3D tracking analysis of
20-min time-lapses,performed on ≥ three islets per time point (± 1 d).
In Vivo Fluorescence Cytolabeling.Under a stereoscope,the needle of a dis-
posable insulin syringe (31-G Ultra-Fine needle;BD) was inserted into the
anterior chamber near the limbus.Great care was taken to avoid contact with
the iris and engrafted islets.Once inside the anterior chamber,the solution to
label islet cells or T lymphocytes was injected slowly over ∼3 to 5 min.Excess
fluid was released periodically through the same entry port to avoid ex-
cessive pressure inside the eye.The slow delivery and small needle gauge
ensured minimal incision and disturbance.To visualize apoptotic cells,10 μL
of undiluted Alexa 568-conjugated annexin V (Invitrogen) and 20 μL of DAPI
(100 μg/mL;Invitrogen) were used.By intentionally damaging the iris with
a needle,we confirmed that positive annexin and DAPI stain was exclusive
to mechanically injured cells.To allow visualization of endocrine and lytic
granules within islet cells and the T lymphocytes,respectively,300 μL of
lysotracker Red DND-99 (5 μM;Invitrogen) were injected while frequently
releasing excess volume.A concentration of 5 μM was used based on the
assumption that the injected volume is further diluted inside the anterior
chamber to a final concentration ≤ 1 μM (41–43).To label different surface
markers on T cells,20 μL of specific antibodies diluted 1:50 in sterile Fc-
blocking solution (SuperBlock Blocking Buffer;Pierce) were directly injected
into the eye as described above.It should be noted that not all infiltrating
cells were labeled because of limited antibody penetration into the tissue.
Used anti-mouse antibodies included,anti–CD8-PE (clone Ly-2),anti-NK 2B4-
PE,anti–CD62L-APC (a.k.a.L-selectin;clone MEL-14) (all from BD-PharMin-
gen),anti–CD4-Alexa 405 (Serotec),anti–CD11a-Alexa 647 (a.k.a.LFA-1;
clone M17/4),and anti–CD25-Alexa 647 (clone PC61;BioLegend).
Statistical Analysis.For statistical comparisons,we used Student t test (to
compare two samples) or one-way ANOVA (to compare multiple samples)
followed by multiple comparison procedures with the Holm-Sidak method
(SigmaStat;Systat).Survival curves were compared using the log-rank test
(Prism;GraphPad) and expressed as median survival time.Data were pre-
sented as means ± SD or SE (SEM).
Additional materials and methods are available in SI Materials
and Methods.
ACKNOWLEDGMENTS.We thank Yelena Gadea,Irayme Labrada,Susana
Villate,Kevin Johnson,Eleut Hernandez,and Diego Espinosa-Heidmann
for technical assistance;Mr.Conrado Freites and Biorep Inc.for custom
fabrications;Drs.Oliver Umland,Over Cabrera,Alberto Pugliese,Jay S.Skyler,
Zhibin Chen,Luca Inverardi,Robert Levy,Martin Kohler,and Stephan Speier
for scientific input;and Dr.George McNamara and the Imaging Core Facility
for technical assistance.Research support was provided by the Diabetes
Research Institute Foundation (A.P.,A.C.,P.-O.B.);National Institutes of
Health/National Institute of Diabetes and Digestive and Kidney Diseases/
National Institute of Allergy and Infectious Diseases Grants 5U19AI050864-10
(to A.P.),1F32DK083226 (to M.H.A.),R03DK075487 and R01DK084321 (to
A.C.),U01DK089538 (to P.-O.B.and A.P.),M01RR16587,5U01DK70460-07,
and R01DK55347-IU42RR016603,General Clinical Research Center (to C.R.),
and Juvenile Diabetes Research Foundation International Grants 4-2004-361
(to C.R.,P.-O.B.,and A.P.) and 4-2008-811 and 17-2010-5 (to C.R.and A.P.).
Additional research support to P.-O.B.was provided through funds fromthe
Karolinska Institutet,the Swedish Research Council,the Swedish Diabetes
Foundation,the Family Erling-Persson Foundation,the Family Knut and Alice
Wallenberg Foundation,the Skandia Insurance Company Ltd.,In Vivo
Imaging of Beta-cell Receptors by Applied Nano Technology (FP7-228933-
2),Strategic Research Programin Diabetes at Karolinska Institutet,the Novo
Nordisk Foundation,and the Berth von Kantzow’s Foundation.
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98(1):77–94.
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after iron nanoparticle labeling.Am J Transplant 8:701–706.
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www.pnas.org/cgi/doi/10.1073/pnas.1105002108 Abdulreda et al.
Supporting Information
Abdulreda et al.10.1073/pnas.1105002108
SI Materials and Methods
Mice.
Animal procedures were performed under protocols
reviewed and approved by the University of Miami International
Animal Care and Use Committee.Mice were purchased fromthe
Jackson Laboratory or bred at our center.Animals were housed in
Virus Antibody Free rooms and kept into microisolated cages
with free access to autoclaved food and water.
Islet Transplantation into the Anterior Chamber of the Eye.
For
metabolic studies,recipient mice were rendered diabetic (non-
fasting glycemia ≥ 350 mg/dL) with a single intravenous strep-
tozotocin injection (200 mg/kg;Sigma).Two groups of diabetic
mice received transplantation into the anterior chamber of one
eye of either ∼300 islet equivalents (IEQ) from syngeneic
(C57BL/6;n =10) or ∼600 IEQfromallogeneic (DBA/2;n =9)
donor mice,respectively.Nonfasting glycemic levels were mon-
itored using portable glucometers (OneTouchUltra2;LifeScan)
to assess graft function (reversal of diabetes) and rejection.
Rejection was confirmed by return to hyperglycemia (defined as
nonfasting glycemia > 200 mg/dL for three consecutive days).
For imaging studies,C57BL/6 (B6.129P2-Cxcr6
tm1Litt
/J;Jackson
Laboratories) (1) mice (n ≥ 11) received ∼100 IEQfromDBA/2
donors into one eye.Control C57BL/6 mice (n = 6) received
∼100 IEQ of syngeneic islets.There was no dependency of the
time to rejection on the alloantigen load (100 versus 600 islets),
as evidenced by similar rejection tempo in metabolic and imag-
ing studies (Fig.1G).
Intravital Microscopy of Transplanted Animals.
Transplanted ani-
mals were anesthetized with an air/isoflurane mixture delivered
through a custom-built mask incorporated in a stereotaxic holder
(SG-4N;Narishige).The mouse was placed on a heating pad and
positioned with the eye containing the engrafted islets facing up.
The eyelid was carefully pulled back as described previously (2,3).
Fluorescence confocal imaging was performed using an upright
Leica DMLFSA microscope with long distance water-dipping
lens (Leica HXC APO20× 0.5W),using sterile saline solution as
an immersion liquid.A digital image of the transplanted eye was
obtained on day 3 after transplantation,and was used as a map
to revisit the same islets for longitudinal imaging.
Islet Transplantation Under the Kidney Capsule.
Islet transplantation
under the kidney capsule was performed in C57BL/6 recipients,as
previously described (4).Briefly,the left kidney was exposed,
a breach was done in the capsule at the cranial pole,and
a polyethylene catheter containing the islets was inserted into the
subcapsular space,the DBA/2 islets released by advancing the
plunger of a 1-mL precision syringe (Hamilton).The incision was
sealed by electric cautery (Medi-Pak),the kidney replaced in the
abdomen,and fascia and abdominal wall sutured.For imaging
studies,DBA/2 islets were transplanted into C57BL/6 (B6.129P2-
Cxcr6
tm1Litt
/J) recipients following the same procedure.
In Vivo Imaging of GFP-Labeled T Lymphocytes Infiltrating Islet
Allografts Under the Kidney Capsule.
Allogeneic islets trans-
planted under the kidney capsule were imaged during rejection
(based on glycemia).Recipient C57BL/6 (B6.129P2-Cxcr6
tm1Litt
/
J) mice were anesthetized as described above.The mouse was
placed on a heating pad and positioned with the side of the
kidney containing the engrafted islets facing up toward the ob-
jective.An incision was carefully made and the grafted kidney
was exteriorized and kept pulled up outside the body without
impeding blood flow using a pair of forceps fitted with soft Tygon
tubing.The exposed kidney was constantly irrigated with sterile
saline solution.For 4D recordings,mice were also injected in-
traperitoneally with xylazine,as described above.In contrast to
the anterior chamber,we were not able to visualize the islet cells
in the kidney using reflected laser due to strong backscatter from
the thick capsular tissue.GFP-labeled T lymphocytes were vi-
sualized through the capsule,albeit,laser penetration and GFP-
fluorescence recovery were limited (Fig.2C).
Immunohistochemistry.
Transplanted mice with GFP-labeled T
lymphocytes (B6.129P2-Cxcr6
tm1Litt
/J) were killed by an iso-
flurane overdose followed by cervical dislocation.Graft-bearing
eyes were procured,fixed in 4% paraformaldehyde (1 h),em-
bedded with sucrose (10%,20%,and 30% in PBS,for 30 min
each),and cryopreserved in O.C.T compound (Tissue-Tek) at
−80 °C.Frozen eyes were cryosectioned (14 μm) perpendicular
to the normal of the iris.Eye sections were washed with PBS (3 ×
10 min) and incubated in Fc-blocking solution (SuperBlock
Blocking Buffer;Pierce) containing 0.1% Triton 100× (1 h).
After permeabilization,sections were incubated overnight in
guinea pig anti-insulin primary antibody (1:500;DAKO) or rat
anti-mouse PE-conjugated antigranzyme B and AlexaFluor 647-
conjugated anti-perforin antibodies (1:500;eBiosciences).Pri-
mary antibody solution was aspirated and goat anti-guinea pig
Alexa 568-labeled secondary antibody (1:500;Invitrogen) was
added overnight.Finally,DAPI (1:600;Invitrogen) was added
for 30 min before washing with PBS (3 × 10 min) and adding
mounting solution (Biomedia Corp.) and glass coverslips.
Elispot Assays for T-Cell Production of IL-2 and IFN-γ.
Elispot plates
(Immunospot M200;Cellular Technologies) were coated with rat
anti-mouse IL-2 (1:250) or IFN-γ (1:500) antibodies (BD Phar-
mingen) and blocked with sterile 1% BSA in PBS.Lymph node
(pooled axillary and cervical) or spleen cells (10
6
per well) from
B6.129P2-Cxcr6
tm1Litt
/J allograft recipient mice were harvested
at day 5 (n = 3) and day 6 (n = 3) after transplantation and
plated in RPMI-1640 media (Invitrogen) with Mitomycin C
(Sigma) treated splenocytes (as stimulators) from DBA/2 mice
(0.5 × 10
6
per well) for 24 h.After washing with PBS 0.025%
Tween 20,plates were incubated with biotin rat anti-mouse or
IFN-γ antibodies (1:250;BD Pharmingen) overnight at 4 °C.
Alkaline phosphatase-conjugated antibiotin antibody (1:250;
Vector Lab) was added for 1.5 h at 20 °C and developed with
nitroblue tetrazolium chloride (1:250;Bio-Rad) and 5-bromo-4-
chloro-3-indolyl phosphatase substrate (Sigma).The revealed
spots were counted on an Immuno-Spot Series 2 Analyzer
(Cellular Technologies).Splenocytes and lymphocytes from non-
transplanted mice (B6.129P2-Cxcr6
tm1Litt
/J;n =3) served as con-
trols.Results are reported as a mean of triplicate wells ± SD.
Flow Cytometry Analysis of Lymphocytes and Splenocytes.
Spleens
and pooled cervical and axillary lymph nodes (ipsi- or contra-
lateral) were harvested from B6.129P2-Cxcr6
tm1Litt
/J allograft
recipient mice at day 6 (n =3 mice) after transplantation,as well
as from nontransplanted control mice (n = 5),and placed into
a sterile Petri dish with a 70-μm nylon cell strainer (BD Bio-
sciences).Cells were collected and treated with TAC lysis buffer
(0.017 M Tris,0.14 M ammonium chloride,pH 7.2) to lyse red
blood cells.The pelleted cells were counted and used for Elispot
assays (see below) and flow cytometry.
Abdulreda et al.www.pnas.org/cgi/content/short/1105002108 1 of 7
Cell suspensions were incubated with Fc-block rat anti-mouse
CD16/CD32 antibody (BDPharmingen) and then stained with rat
anti-mouse CD4-APC and rat anti-mouse CD8-PerCP Cy5.5
antibodies (eBiosciences).After staining,cells were incubated
with DAPI to identify nonviable cells.Cell-surface markers on
stained cells were analyzed by flow cytometry by gating on the
DAPI-negative and GFP-positive cells and collecting ∼50,000
events.Data were acquired on a BD LSRII Special Order Sys-
tem (BD Biosciences) and analyzed using FACS Diva 6.0 anal-
ysis software (BD Biosciences).
TAK-779 and Chemokine Treatments.
TAK-779 (N,N-dimethyl-N-
(4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-Benzocyclohepten-8-yl]
carbonyl]amino]benzyl)-tetrahydro-2H-Pyran-4-aminium chlo-
ride) was obtained through the AIDS Research and Reference
Reagent Program,Division of AIDS,National Institute of Al-
lergy and Infectious Diseases,National Institutes of Health.
TAK-779 solution was prepared in saline at 1.25 mg/mL and
administered either systemically [250 μg daily between post-
operative day (POD) +10 and POD +17;intraperitoneally] or
directly into the anterior chamber of the eye (50 μM;15–20 μL
per injection).The mix of CXCL9 (100 nM;R&D Systems) and
CXCL10 (100 nM;Biosource) was prepared in PBS.For direct
injection into the anterior chamber of the eye of anesthetized
mice,TAK-779 or mixed CXCL9 and CXCL10 solutions were
loaded into separate Tygon tubing connected to 500-μL Hamil-
ton syringes with screw-driven plungers.Glass micropipettes
(∼30-μm tip diameter) were back-filled after connecting to the
tubing and mounted onto separate xyz micromanipulators
(Narishige).Injection through the cornea was done slowly to
minimize turbulence.
1.Unutmaz D,et al.(2000) The primate lentiviral receptor Bonzo/STRL33 is coordinately
regulated with CCR5 and its expression pattern is conserved between human and
mouse.J Immunol 165:3284–3292.
2.Speier S,et al.(2008) Noninvasive in vivo imaging of pancreatic islet cell biology.Nat
Med 14:574–578.
3.Speier S,et al.(2008) Noninvasive high-resolution in vivo imaging of cell biology in the
anterior chamber of the mouse eye.Nat Protoc 3(8):1278–1286.
4.Pileggi A,et al.(2005) Prolonged allogeneic islet graft survival by protoporphyrins.Cell
Transplant 14:85–96.
Abdulreda et al.www.pnas.org/cgi/content/short/1105002108 2 of 7
Fig.S1.Intraocular transplantation enables longitudinal in vivo studies in the same islet grafts noninvasively.(A) H&E stain of a frozen section of a whole
mouse eye showing an islet engrafted on top of the iris in the anterior chamber (ACE) of the eye.(Scale bar,200 μm.) (B) Repeated images of the same
islet allograft (outlined) showing that its progressive destruction was paralleled by increasing T-cell (green) infiltration.Focal islet destruction occurred near
clusters of ruffled T lymphocytes.(Lower) Close-ups of boxed areas.[Scale bars,100 μm(Upper),50 μm(Lower).] (C) Islet allograft destruction correlated well
with T-cell infiltration (each circle represents a single islet).(D) Longitudinal changes in the volume of individual islets.Islets were deemed rejected after ≥ 30%
reduction in initial islet volume because no more than 13%(i.e.,mean + SD) decrease in the volume of syngeneic islet grafts was observed.(E) Longitudinal
imaging of the same islet (outlined) revealed incipient revascularization (red) at 5 d after transplantation.Initially,the majority of infiltrating GFP-labeled T
lymphocytes (green) appeared near iris blood vessels outside the islet (days 5 and 10).As vessels grew further within the islet,intraislet T-cells appeared near
newly formed vessels (day 17).Blood vessels were visualized by intravenous injection of TRITC-labeled dextran (2 × 10
6
D).(Scale bar,100 μm.) (F) Longitudinal
dynamic analysis of displacement (traveled distance in straight line) and path length (cumulative traveled distance) during progression of islet allorejection.
Both parameters increased significantly at onset of acute rejection (day 0) compared with ≤ day −3 (shown as means ± SEM;*P < 0.05).
Abdulreda et al.www.pnas.org/cgi/content/short/1105002108 3 of 7
Fig.S2.Intraocular transplantation enables in vivo immunocytolabeling in situ.(A) Graft-infiltrating GFP-labeled T cells were immunoreactive to antibodies
against CD25 and LFA-1 injected directly into the ACE.(Scale bar,5 μm.) (B) Flow cytometry analysis gated on viable GFP-labeled lymphocytes in cervical and
axillary lymph nodes derived from intraocular islet allograft recipients at POD +5.(C) Immunostaining of fixed intraocular islet allograft confirmed the
presence of granzyme B and perforin in graft-infiltrating T lymphocytes (green) and showed a staining pattern similar to the in vivo results.Shown images are
representative of a minimum of triplicate experiments.(Scale bar,10 μm.) (D) Snapshots of a 3D time-lapse recording showing allograft-infiltrating ruffled T-
cells (green) interacting with one another within dynamic clusters (Movie S7).A representative cell (outlined in red) contacted at least one other T-cell (outlined
in gray) at any given time point.By conservative estimate,the selected cell contacted a minimumof 5 cells in the selected frames of this 20 min recording while
moving within a confined space (track highlighted in white in the last frame).Images shown as maximum projections.(Scale bar,20 μm.) (E) Instantaneous
velocity and 3D shape index (surface area/volume) of ruffled T-cells within allograft (POD +12) are significantly different from those of round and elongated
cells.The higher 3D shape index is likely to be due to the complex dynamic behavior of ruffled cells as they engage in simultaneous contacts with neighboring T
cells and target islet cells.Data presented as means ± SEM(results based on 161 images of 18 round T cells,203 images of 27 elongated cells,and 896 images of
32 ruffled cells;*P < 0.05).(F) As a measure of randomness and confinement in cellular motility,despite increased translational movement during acute
rejection,we plotted the mean displacement or chemotactic index (displacement/path length) vs.time (1).This showed increased confinement and random
migration by ruffled T cells compared with elongated ones (n = 137 ruffled and 26 elongated cells).Based on our results during acute rejection,the mean
velocity of graft-infiltrating T lymphocytes (with > 60%being ruffled;see Fig.2F) was ∼30%of that in the lymph node (1,2).Assuming a similar reduction in
the motility coefficient within the islet allografts compared with the lymph node (3),a value of ∼20 μm
2
/min is expected;we calculated however an average
value of ∼8 μm
2
/min,which further supported increased confinement in the movement of the predominating ruffled T-cells within allografts during rejection.
1.Cahalan MD,Parker I (2008) Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.Annu Rev Immunol 26:585–626.
2.Miller MJ,Wei SH,Cahalan MD,Parker I (2003) Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.Proc Natl Acad Sci USA 100:2604–2609.
3.Worbs T,Mempel TR,Bölter J,von Andrian UH,Förster R (2007) CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo.J Exp Med 204:489–495.
Abdulreda et al.www.pnas.org/cgi/content/short/1105002108 4 of 7
Fig.S3.Reduced fraction of intraislet ruffled T-cells associates with slower islet destruction.(A) Relative proportions of the different T-cell phenotypes before
and after CCR5/CXCR3 systemic and chronic blockade with TAK-779 in the pre- and during/post rejection periods.Data are presented as stacked bars (light
color,untreated;full color,treated).The proportion of ruffled cells was reduced significantly with TAK-779 treatment compared with nontreated animals (P <
0.05).(B) The rate of islet destruction was diminished in treated mice.To compare the rejection kinetics during the acute phase,we used as reference the onset
of effective rejection defined as islet destruction beyond ∼13%,which was the reduction (mean + SD;gray zone) in the volume of syngeneic grafts.The slope
of the regression lines (dashed lines) fit to the data points was significantly less steep in TAK-779 treated animals,indicating slower kinetics of islet destruction.
Results were pooled from15 islets (TAK-779 treated;three mice) and 22 islets (untreated;five mice) and presented as means ± SEM[n = 4–15 islets per group
(A);and n = 15 islets (TAK-779 treated) or 5–31 islets (untreated) per time point (B)].*P < 0.05.
Movie S1.Time-lapse recording (20 min) of an intraocular islet (outlined) showing migration of graft-infiltrating GFP-labeled T lymphocytes (green) at onset
of rejection (POD +21).The islet,engrafted on the iris,is readily visualized by laser reflection (back-scatter).The second part of the movie shows a 3D rendering
of the islet to illustrate the dynamic behavior of T-cells within the graft.Inside the islet,a movement trajectory (arrowhead at end) of a fast-moving T-cell
(highlighted in white) is shown.Other T-cells within the graft become evident as the reflection channel (gray) is masked.(Scale bars,90 μm.)
Movie S1
Movie S2.Repeated 20-min time-lapse recordings (shown as maximumprojection) of the same intraocular islet allograft (gray) during the different phases of
rejection (based on ≥ 30%loss in islet volume).The graft was relatively intact at POD +18 (early phase).By POD +21,loss in islet cell mass/volume was evident
(acute phase) and coincided with predominance of ruffled T-cells (green) engaging in highly dynamic clusters within the islet.At POD +24 (late phase),only
remnants of the islet were visible.The immune attack was restricted to the allograft with no apparent damage to surrounding/underlying iris tissues.(Scale
bars = 90 μm.)
Movie S2
Abdulreda et al.www.pnas.org/cgi/content/short/1105002108 5 of 7
Movie S3.Fluorescence-labeled anti-CD4 (blue) and anti-CD8 (red) antibodies were injected into the anterior chamber of the eye of an allogeneic islet re-
cipient at POD+7.Confocal images were acquired during a 20-min time-lapse recording (shown as maximumprojection) that was started ∼5 min after injection.
(Scale bars,90 μm.)
Movie S3
Movie S4.Three-dimensional rendering of a recording (20 min) of an intraocular islet allograft (gray;POD +19) demonstrating preferential localization of
ruffled T lymphocytes (green) near dead/dying islet cells.Apoptotic islet cells were visualized by intraocular injection of annexin V (red) and DAPI (blue).A few
round T-cells surrounding the islet showed no direct association with islet cells,and elongated cells migrated away from the islet.By contrast,ruffled cells
closely associated with annexin V and DAPI-positive islet cells.
Movie S4
Movie S5.Time-lapse recording (10 min) acquired during rejection (POD +19) within an islet allograft (gray) showing lack of polarization of lytic granules
within infiltrating ruffled T-cells (green) toward single contact zones with target islet cells.Lytic granules were visualized with lysotracker (red) injected into
the ACE.At the end of the movie,a close-up view(in 3D) of the boxed area shows ruffled T-cells contacting multiple islet cells as they maintain net translational
movement,as evidenced by their movement tracks.(Scale bars,30 μm.)
Movie S5
Abdulreda et al.www.pnas.org/cgi/content/short/1105002108 6 of 7
Movie S6.Three-dimensional renderings of the round,elongated,and ruffled T-cell phenotypes shown in Fig.2.The movie demonstrates the spherical shape
of round cells and the typical elongated shape of fast-moving cells with a characteristic large leading edge and a thin,long europod.It also shows a cluster of
ruffled T-cells displaying irregular shapes characterized by long cytoplasmic processes extended in multiple directions.
Movie S6
Movie S7.Three-dimensional rendering of the snapshots shown in Fig.S2D.The movie highlights a cluster of ruffled T-cells in an area where islet tissue was
rejected and shows the complex dynamic behavior of the cells.
Movie S7
Movie S8.Sequential recordings (10-min each) in the same intraocular islet allograft (POD +22) before and after intraocular injection of TAK-779 or a CXCL9/
CXCL10 mix.Fluorescence confocal images are shown as maximum projections in intensity scale to illustrate phenotypic changes in the graft-infiltrating GFP-
labeled T lymphocytes.Notice the change in the cellular morphology and motility near and within the graft (outlined) after TAK-779 or CXCL9/CXCL10 ad-
ministration.With TAK-779 treatment,the cells converted to the stationary,round phenotype which was evidenced by the reduced intensity (blue) within their
notably shorter lamellipodia around the bright (red) cell bodies.Such changes were reversed with subsequent administration of CXCL9/CXCL10.Recordings
were acquired ∼10 min after each treatment;during which time,additional cells migrated into the imaged field.This finding was especially evident after
CXCL9/CXCL10 treatment increased movement which associated with significantly increased cellular motility (Fig.3B).(Scale bars,10 μm.)
Movie S8
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