Title Page
Impact of LFA-1 blockade on endogenous allo-specific T cells to multiple mH-Ag mismatched
cardiac allograft
Jean Kwun1,3,5*, Alton B. Farris2, Hyunjin Song1, William T. Mahle3, William J. Burlingham4 and Stuart J.
Knechtle1,3,5*
Abstract: 223 words; Text: 2967 words; References: 34; Figures: 5; Color Figures: 4; Tables:0
*Address for correspondence: Jean Kwun, PhD, 207 Research Drive, 313
Jones Building, Durham, NA 27710, U.S.A. Phone: 404-775-9782; Fax: 404727-3660; E-mail: jean.kwun@duke.edu
or Stuart J Knechtle, MD, 330 Trent Drive, DUMC Box 3512, Durham, NC
27710, U.S.A. Phone: 919-613-9687; Fax: 919-684-8716; E-mail:
stuart.knechtle@dm.duke.edu (JK and SJK share last authorship for this work)
1
Footnotes
1. Footnotes to the Title:
1. Author contributions: J.K. participated in research design, performed surgical procedures,
experimental animal care, conducted in vitro experiments, interpreted data, prepared the
manuscript, and funded the research; A.B.F. interpreted data (pathologist) and prepared the
manuscript; H.S. participated in performing in vitro experiments (including histology and IHC),
prepared the manuscript; W.T.M. interpreted data and prepared manuscript; W.J.B. participated
in research design, interpreted data and prepared manuscript; S.J.K. participated in research
design, interpreted data, prepared manuscript and funded the research.
2. Supports: This work was supported by: Cardiovascular Biology Pilot Funding from the Center
for Cardiovascular Biology (CCB), Children’s Healthcare of Atlanta (CHOA) and the University
Research Committee (URC) Grant from Emory all awarded to J.K. This work was partially
supported by grant the Mason Chair Grant of CHOA awarded to S.J.K.
3. Disclosures: The authors declare no conflicts of interest
2. Footnotes to the Author’ names:
1. Emory Transplant Center, Department of Surgery, Emory University School of Medicine, Atlanta,
GA 30322
2. Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322
3. Department of Pediatrics, Emory University School of Medicine, and Center for Cardiovascular
Biology, Children’s Healthcare of Atlanta, Atlanta, GA30322
4. University of Wisconsin-Madison, Department of Surgery, Division of Transplantation, 600
Highland Avenue, CSC H4/733, Madison, Wisconsin 53792
5. Duke Transplant Center, Department of Surgery, Duke University Medical Center, Durham, NC
27710
2
Nonstandard Abbreviations
AR, Acute rejection; ACR, Acute cellular rejection; CAV, Cardiac allograft vasculopathy; CFSE,
Carboxyfluoresceinsuccinimidyl ester; CR, Chronic rejection; CsA, Cyclosporine A; CTL, cytotoxic T
lymphocyte; DTH, delayed type hypersensitivity; ELISpot, enzyme-linked ImmunoSPOT; GIL, Graft
infiltrating lymphocyte; IFN-, Interferon-gamma; LFA-1, Leukocyte Function-associated Antigen-1;
mAb, monoclonal antibody; mH-Ag, minor histocompatibility antigen; MMF, Mycophenolate mofetil;
PBMC, Peripheral Blood Mononuclear Cells; PML, Progressive Multifocal Leukoencephalopathy; POD,
Post Operation Day; PTLD, Posttransplant Lymphoproliferative Disease; TCR, T-cell Receptor
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Abstract
Background. Blocking LFA-1 in organ transplant recipients prolongs allograft survival. However, the
precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic
rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the pre-eminent cause of late
cardiac allograft failure characterized histologically by concentric intimal hyperplasia.
Methods. Anti-LFA-1 mAb was used in a multiple minor Ag mismatched, BALB.B (H-2b) to C57BL/6
(H-2b), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using MHC
multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags.
Results. LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced
CD8 T cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T
cell expansion rather than less trafficking. LFA-1 blockade significantly suppressed the clonal expansion
of mH-Ag-specific CD8 T cells during the expansion and contraction phase. CAV development was
evaluated with morphometric analysis at POD 100. LFA-1 blockade profoundly attenuated neo-intimal
hyperplasia (61.6 vs. 23.8%; p<0.05), CAV affected vessel number (55.3 vs. 15.9%; p<0.05), and
myocardial fibrosis (grade 3.29 vs. 1.8; p<0.05). Finally, short-term LFA-1 blockade promoted long-term
donor-specific regulation, which resulted in attenuated transplant arteriosclerosis.
Conclusions. Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T cell expansion
and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with antiLFA-1 rather than long-term treatment.
Keywords: LFA-1, Minor antigen; Cardiac allograft vasculopathy; Tetramer; Tolerance
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Introduction
Stable organ transplant function depends on the control of T cell immune responses by pharmacologic
immunosuppression. Despite the success of immunosuppression in reducing acute rejection (AR) and
early graft loss in clinical transplantation, chronic rejection (CR) is still an impediment to long-term graft
survival. There remains an urgent need to develop alternative strategies to control rejection in
transplantation, as conventional immuosuppression regimens which are mostly based on nephrotoxic
calcineurin inhibitors (CNIs), may contribute to chronic rejection.
Leukocyte function-associated antigen 1 (LFA-1, 2-integrin, CD11a/CD18) is expressed on the surface
of all white blood cells (1) and has been identified with several distinct functions. LFA-1 is involved in
the arrest of rolling leukocytes on target endothelium which is crucial for lymphocyte homing to lymph
nodes or inflamed tissues (2). LFA-1 plays an important role in the conjugation of a T cell binding to its
APC, which is necessary for an efficient T cell activation. It is also believed that the signal produced by
LFA-1 promotes T cell activation and differentiation (3). Due to its associated role in T cell trafficking
and activation (4-7), monoclonal antibody targeting LFA-1 has been applied in various transplant models
and showed donor specific tolerance (8, 9) or anti-graft transmigration (10, 11). However, the
involvement of endogenous allo-specific T cells in this process has not been fully addressed. Therefore,
we investigated the efficacy of LFA-1 blockade in established multiple minor Ag mismatched cardiac
allograft model allowing visualization of allo-specific T cells. Our current study shows that anti-LFA-1
mAb ameliorates acute and chronic rejection by abolishing initial allo-specific CD8 T cell expansion and
permitting long-term regulation of allo-specific T cells.
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Results
LFA-1 blockade prevents early acute rejection and graft dysfunction
Heterotopic heart transplants were performed in C57BL/6 (H-2b) mice, using BALB (H-2b) mice as
donors. In the fully MHC-mismatched combination (BALB/c into C57BL/6) heart grafts were rejected
promptly within 10 days (12). In contrast, the multiple minor histocompatibility Ag (mH-Ag)-mismatched
combination (BALB into C57BL/6) showed either delayed AR (median 14 days) or spontaneous longterm allograft survival (Figure 1A). All transplanted BALB hearts showed weakening of the heartbeat
between 7 and 14 days after transplantation, indicative of an AR episode. The heartbeat score of most
long-surviving grafts remained low (1+ to 2+), while in others it regained strength up to a 3+ level
(Figure 1B). Anti-LFA-1 mAb was administered i.p. on day 0, 1, 7 and 14 post-transplant. Surprisingly,
LFA-1 blockade eliminated AR (Figure 1A) and resulted in a better heartbeat quality than that of the
untreated recipients at day 100 (Figure 1B). Allografts explanted at POD10 showed lack of mononuclear
cells infiltration with anti-LFA-1 mAb treatment (Figure 1C). During the AR phase, the most prominent
subsets of the perivascular and myocardial infiltrate were CD8 T cells that were dramatically reduced
after anti-LFA-1mAb treatment (Figure 1C). In conclusion, LFA-1 blockade induces more significant
allograft prolongation by reducing CD8 T cell infiltration into the graft.
LFA-1 blockade profoundly suppresses immunodominant H60-specific CD8 T cell expansion
Interestingly, GIL had a much higher percentage of CD8 T cells compared to corresponding PBMCs or
spleen lymphocytes. Based on T cell infiltration patterns shown in figure 1, we hypothesized that the
proportional reduction of CD4 T cells in the graft must be secondary to increased CD8 T cell graft
infiltration. To evaluate the effect of LFA-1 blockade in T cell trafficking, CD4 and CD8 T cells in the
different immune compartments were evaluated at POD10. A significant reduction of CD8 GIL was
found in anti-LFA-1 mAB treated animals (p<0.05) compared to the untreated controls. However,
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retention (or lymphocytosis) of these cells in spleen and peripheral blood was not observed (Figure 2A).
Since overall CD4/CD8 T cells do not represent antigen-specific T cell expansion, we then tracked down
donor Ag-specific T cells with tetramers. We previously reported that H60-specific CD8 T cells showed
eminent immunodominance in BALB.B to B6 heart transplantation (13, 14). As shown in figure 2B,
peripheral H60-specific CD8 T cells from untreated animals (POD10) demonstrated homing
(CD11ahiCD44hiCD62Llo) and chemokine (Cxcr3+Ccr5+Ccr7-) receptor expression of a traditionally
activated Th1-related cell phenotype. Through the combination of this homing/chemokine receptor
expression, these cells are immunophenotypically ready to leave the secondary lymphoid organ and get to
the inflammatory site. To confirm their role of the mH-Ag specific CTLs in AR, we investigated their in
situ localization in heart grafts. The heart sections of H60 CTLs were visualized with confocal laserscanning microscopy (CLSM). H60 tetramer+CD8+ CTLs were detected in POD 10 untreated recipients
during AR (Figure 2C). Finally, allo-specific CD8 T cells were evaluated in spleen, blood, and graft with
H60 tetramer staining. While H60-specific CD8 T cells demonstrated a selective accumulation in the graft,
surprisingly, H60 tetramer+ allo-specific CD8 T cells were significantly reduced in all immune
compartments after LFA-1 blockade (Figure 2D). The significant reduction of H60 tetramer+ CD8 T cells
was also observed in the draining lymph nodes after LFA-1 blockade, while the CD8 T cell proportion
between the treated and untreated groups was not changed. Interestingly, CD4 T cell proportion was
significantly decreased after LFA-1 blockade (Supplemental figure 1). Co-immunodominant H4 mHAg specific CD8 T cells also showed similar patterns (Supplemental figure 2). These data suggest that
LFA-1 blockade prevents allo-specific T cell expansion rather than their transmigration during the AR
phase.
LFA-1 blockade suppresses clonal expansion of all immunodominant CD8 T cells.
Even though H60 specific CD8 T cells are the most immunodominant and prevailing donor-specific CD8
T cell population after heart transplantation, there are co-immunodominant minor antigens in this
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combination, particularly the H4 and H7 mH-Ags (14). To rule out the possibility of immunodomiance
alteration with LFA-1 blockade, co-immunodominant H4, H7, H13, and H28 mH-Ag specific CD8 T
cells were evaluated. However, we found that expansion of these immunodominant mH-Ag specific CD8
T cells in BALB.b to B6 combination were inhibited (Figure 3A). Co-immunodominant H4 and H7
specific CD8 T cells were significantly reduced with LFA-1 blockade (Figure 3B). H13 and H28 specific
CD8 T cells did not show a significant reduction, but this was secondary to minimal expansion with heart
transplantation in both treated and untreated groups. In order to evaluate the overall anti-donor response,
we measured IFN- producing T cells with ELISPOT from both untreated and anti-LFA-1 mAb treated
animals at day 10 against donor splenocytes (direct) or donor splenocyte sonicates (indirect). As expected,
the frequency of donor-specific alloreactive IFN--producing cells in the minor mismatched combination
was significantly smaller than that of the major mismatched combination from both direct (220 ± 13.58 vs.
1216 ± 252.9; p = 0.017) and indirectly (143.3 ± 26.47 vs. 304.3 ± 24.52; p=0.011) restimulated
splenocytes. As shown in figure 3C, the elevated frequency of allo-reactive IFN- producing cells during
AR was significantly reduced by LFA-1 blockade. The limited number of allogeneic T cells in this system
might be the main mechanism of AR prevention, therefore these data suggest that LFA-1 blockade
prevents AR by suppressing the expansion of donor reactive effector T cells.
LFA-1 blockade prevents CAV development in multiple minor Ag mismatched model
To determine the status of prolonged cardiac allografts, transplanted BALB heart was explanted on
POD100. The representative elastic trichrome stain from untreated animal demonstrated patchy interstitial
fibrosis, subepicardial fibrosis, and elastic layers of the aorta that were surrounded by a prominent
inflammatory infiltrate. On the other hand, an elastic trichrome stain with anti-LFA-1 mAb treatment
highlighted the intact elastic layers of the aorta and showed no evidence of interstitial fibrosis (Figure
4A). To quantify CR, we performed morphometric analysis for neointimal hyperplasia, number of
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diseased vessels, and degree of fibrosis. The degree of luminal occlusion was 61.6 ± 6.6% for untreated
recipients and 23.8 ± 7.6% for anti-LFA-1 mAb treated recipients (p < 0.05). Both diseased vessel number
of individual grafts (55.3 ± 7.4 vs. 15.9 ± 5.0%) and the degree of fibrosis (3.29 ± 0.4 vs. 1.8 ± 0.6) were
significantly decreased in anti-LFA-1 mAb treated recipients. (p<0.05; Figure 4B). This could be simply
due to the lack of AR associated injuries with anti-LFA-1 mAb treatment, however, ongoing CD4 and
CD8 T cell graft infiltration was decreased (Figure 4C). Both cell-mediated and antibody-mediated
responses contribute to CAV development (15). To identify the relative importance of antibody-mediated
rejection in CAV development, we evaluated C3d deposition in C57BL/6 recipients of BALB.B hearts.
Compared to the untreated controls, anti-LFA-1 mAb treatment resulted in the reduction of C3d
deposition in both day 10 and day 100 post-transplantation (Supplemental figure 3). Unfortunately, there
is no means to track down the donor specific antibodies in the non-MHC DSA to confirm the involvement
of AMR in this CAV development. Currently, we are testing anti-LFA-1mAb in a murine CAMR model
(16). These data suggests that abolishing mH-Ag-specific CD8 T cell expansion by anti-LFA-1 mAb
prevents not only acute allograft rejection but also the development of chronic allograft vasculopathy.
LFA-1 blockade induces long-term donor-specific regulation
Less CAV with T cell graft infiltration represents either lack of anti-donor response or on-going donorspecific regulation. To further examine the existence of regulation or hyporesponsiveness in cardiac
recipients, DTH responses in recipients and generation of continuous effector CD8 T cells were evaluated.
As illustrated in Figure 5A, anti-LFA-1 mAb treated recipients only developed background swelling
responses when challenged with donor (BALB) derived splenocytes at POD 100. However, untreated
recipients mount DTH responses. This could also represent the less efficient tissue homing of allospecific T cells. We also evaluated the conventional T reg (CD4+CD25+FoxP3+ T cells) cells in the
recipients as well as functionally known T reg phenotypes that could expresses TB21, LAP, GITR. None
of these populations were proportionally increased in anti-LFA-1 mAb treated animals (Data not shown).
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However, even though the proportion of T regs (CD4+CD25+FoxP3+) showed similar levels in untreated
and anti-LFA-1 treated animals at day 100 (Figure 5B), increased T regs were identified in anti-LFA-1
mAb treated long-term recipients after 48 hours of restimulation with donor (BALB.B) T cells (Figure
5B). Furthermore, the splenocytes from the anti-LFA-1 mAb treated recipients actively suppressed the in
vitro proliferation of allo-reactive T cells in MLR (Figure 5C). These data suggest that initial short-term
LFA-1 blockade promotes long-term donor-specific regulation.
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Discussion
This study was undertaken to evaluate the efficacy of LFA-1 blockade longitudinally in AR and later CR
development since these two responses are closely related. Here, we used multiple minor Ag mismatched
heart transplantation because the murine minor H antigen is well characterized, and recently developed
MHC multimers allow us to track the donor specific CD8 T cells. In addition, the BALB to C57BL/6
combination models acute and chronic rejection without any immunosuppressive manipulation, thus
providing a platform to test the efficacy of anti-LFA-1 mAb monotherapy for preventing both types of
rejection. This is unlike fully MHC mismatched combinations (BALB/c to C57BL/6), which show no
spontaneous acceptance and provide no means of tracking down allo-specific T cells. H60 mH-Ag
specific CD8 T cells have shown immunodominance among mH-Ags after donor splenocyte transfusion
(17, 18) and bone marrow transplantation (19-22). In this study, we identified dominant (H60) and
subdominant (H4 and H7) MHC class I-restricted mH-Ag-specific CD8 T cells after cardiac
allotransplantation. Interestingly, anti-LFA-1 mAb treatment completely prevented AR and significantly
reduced graft infiltrating CD8 T cells (Figure 1). LFA-1 (2-integrin) may play an important role in graft
rejection, mediation of infiltration into the graft, and dissemination of the antigenic message to the
lymphoid tissues of the host. In accordance with this, researchers found increased peripheral blood
leukocyte counts after anti-LFA-1 treatment (5,6). However, we have not found any fluctuation of CD4
and CD8 T cell proportion in other immune compartments (Figure 2A). Furthermore, the most
immunodominant H60 mH-Ag specific CD8 T cells as well as co-dominant H4 and H7 mH-Ag specific
CD8 T cells were significantly reduced in all immune compartments with anti-LFA-1 mAb treatment
(Figure 2 and 3). LFA-1 blockade reduced in vitro anti-donor Th1 response back to baseline levels
(Figure 3B). This suggests that LFA-1 blockade suppresses initial endogenous allo-specific T cell
expansion. However, this is not a definitive conclusion as this model induces only an oligoclonal T cell
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activation. It is possible that the polyclonal T cell activation with a full MHC disparity would demonstrate
the anti-migration effect in addition to anti-activation effect.
We also found a dramatic decrease of neo-intimal hyperplasia and fibrosis compared to the untreated
controls (Figure 4). Recently, donor specific antibody (DSA) has been suggested as a contributing
factor to chronic rejection. Therefore, it is reasonable to consider that LFA-1 plays a critical role in
mediating humoral responses (23, 24). Initial cognate T-B cell interaction could be blocked by antiLFA-1 mAb and thereby suppress down-stream humoral responses. We observed reduced C3d
deposition from the graft that supports this conceptually. However, the minor antigen mismatch
model does not allow us to evaluate DSA since most mH-Ags are not expressed on the surface. It is
interesting that a short course of anti-LFA-1 mAb treatment induced long-term suppression of allospecific T cells and directly affected on CAV development at day 100. One could argue that this is due to
the lack of AR in anti-LFA-1 mAb rather than a continuous process. However, it is surprising to see that
both CD4 and CD8 T cell infiltration was greatly reduced. This suggests that there is an intact stable
regulation related to CAV development. Lack of DTH response confirmed a donor-specific regulation in
anti-LFA-1 mAb treated animals (Figure 5A). Furthermore, splenocytes from anti-LFA-1 mAb treated
recipients suppressed a T cell proliferation from the untreated recipient splenocytes against donor
stimulation in vitro (Figure 5C), further suggesting active long-term anti-donor regulation. Recent efforts
to evaluate the immune synapse might give some insight into the mechanism of graft prolongation and
tolerance following therapy targeting the LFA-1 molecule. Even though LFA-1 is not directly involved in
the immune synapse, it provides a scaffolding structure and stabilizes the immune synapse (25, 26). Thus
perturbation of LFA-1 structure can lead T cell inactivation. Furthermore, this non-ideal immunogenic
condition can promote naïve T cells differentiation into regulatory T cells (27). Therefore, continuous
stimulation with allo-antigens in a depleted LFA-1 signal environment (or lack of other signal without
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support of LFA-1) may lead to the induction of specific tolerance (Figure 5B). Taken together, LFA-1
blockade may inhibit alloreactive T cell expansion and induce more regulation. Such a mechanism
supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment designed as
an anti-trafficking strategy. This approach to short-term (induction) use of LFA-1 blockade differs from
the human clinical trial approach previously published (28, 29).
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Material and Methods
Mice
Male BALB/c (H-2d), BALB (H-2b) and C57BL/6 (H-2b) mice, 6-8 weeks of age, were purchased from
The Jackson laboratory (Bar Harbor, ME). All mice were housed in plastic cages with controlled
light/dark cycles and provided ad libitum with food and water in Emory University animal resources
(Atlanta, GA). All mouse experiments were performed in accordance with the guidelines and compliance
of the institutional Animal Research Ethics Committee.
Heart transplantation
Heart transplantation was performed using a modification of the methods described previously (14). The
grafts were monitored by daily palpation and graded from 4+ (strongest beat) to 0 (no beat), and rejection
was confirmed by laparotomy. Animals were either untreated or treated with anti-LFA-1 mAb (KBA; rat
IgG2a) 200g, i.p. on POD 0, 1, 7 and 14.
Histology and Immunohistochemistry
The explanted hearts underwent serial sectioning (5m). H&E stains were performed for routine
examination. Elastic trichrome staining was performed for morphometric analyses of arterial intimal
lesions as previously reported (16). Frozen sections were cut (5m), fixed in 4% paraformaldehyde, and
blocked with a 10% BSA (Sigma, MO) for 1 hour, followed by 5% non-fat milk. Sections were then
incubated at 4oC with the primary antibody (anti-CD4 (L3T4) or anti-CD8 (Pharmingen, CA)) and
detected using an anti-rat or mouse HRP-Polymer kit (Biocare medical, CA). In situ tetramer staining was
performed on 5m thick cryosections (30) and 200m thick unfixed vibroatome sections (31, 32) as
described previously. The sections were blocked with a 10% BSA followed by 5% non-fat milk.
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Tetramers were added at a concentration of 0.5g/ml and incubated overnight at 4oC. Sections were
washed, and an amplification step was performed using Cy5 conjugated rabbit -PE (biomed) diluted
1:100 dilution. After incubating 30 mins, tissue sections were washed and fixed with 4%
paraformaldehyde for 10 mins. Finally, rat anti-mouse CD8 was applied and incubated 60 minutes and
subsequently visualized with a fluorescent microscope and Bio-Rad 1000 confocal microscope (Bio-Rad).
ELISPOT assay
An IFN- ELISPOT assay was performed as described previously (33). Briefly, unifilter 96 well plate
(Whatman) were coated with anti-mouse IFN- mAb (5 g/ml; BD Bioscience). Splenocytes from
untreated or anti-LFA-1 mAb treated B6 recipients (4 x 105/well) were plated with equal number of
irradiated (2000rad) BALB.B splenocytes (Direct response) or 1x105 allogeneic peptide (BALB.B
splenocyte sonicates) pulsed autologous DC (Indirect response). For allo-specific response of full MHC
mismatch, irradiated BALB/c splenocytes or BALB/c splenocyte sonicates were used. After incubation at
37°C with 7% CO2 for 48hrs, a secondary Ab (2 g/ml; BD Biosciences) was added then developed and
enumerated using an ImmunoSpot analyzer (AID, Strassberg).
Flow cytometry
Briefly, cell suspensions were resuspended in 2% FBS, 0.2% Sodium azide PBS and were stained with
Biotin, PE, FITC, PerCp, or APC conjugated antibodies directed at mouse CD3 (145-2C11), CD4
(H129.19), CD8 (53-6.7), CD11a (2D7), CD28 (37.51), CD44 (IM7), CD62L (MEL-14), , Ccr5 (HMCcr5), Ccr7 (4B12), and isotype controls (BD Pharmingen, San Diego, CA). PE-conjugated H60 mH-Ag
(LTFNYRNL) Tetramer (Beckman Coulter, CA) or H4b (SGIVYIHL), H7b (KAPDNRDTL), H13b
(SSVIGVWYL), H28 (FILENFRPL) Pentamers (Proimmune, UK) was co-stained. Cytometric analysis
15
was performed using a FACS Caliber cytometer (BD Bioscience, San Jose, CA) and analyzed using
FlowJo (Tree Star, San Carlos, CA) software.
Direct in vivo DTH assay
In order to measure delayed type hypersensitivity (DTH) against donor antigens, 10x106 of BALB.B
donor splenocytes (in 40l DPBS) were injected directly to footpad of untreated (n=3) or anti-LFA-1
mAb treated recipients (n=3). Swelling of the footpad was measured blindly 24 hr later, with values
compared with the thickness before injection.
CFSE proliferation assay
Splenocytes were recovered from untreated (day 10) and anti-LFA-1 mAb treated (day 100) recipients
and labeled with CFSE for 15 and 10 minutes, respectively. Recipient driven splenocytes were directly
co-cultured with irradiated BALB.B splenocytes or mixed as designated ratio (1:16 ~16:1) for 4 days.
Cultured cells were stained for CD3 and analyzed by flow cytometry. The gated CD3+ T cells were
analyzed for CFSE decay in the FL1 channel to assess proliferation.
Statistical analysis
Experimental results were analyzed by a GraphPad Prism (GraphPad Software 6.0a, San Diego, CA). The
log-rank test for differences in graft survival and Mann-Whitney nonparametric test were used for other
data. All the data are presented as mean ± SEM unless designated in figure legend. Values of p less than
0.05 were considered as statistically significant.
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Acknowledgments
This work was supported by the grants awarded to J.K.: Cardiovascular Biology Pilot Funding from the
Center for Cardiovascular Biology (CCB), Children’s Healthcare of Atlanta (CHOA) and the University
Research Committee (URC) Grant from Emory university. This work was partially supported by the grant
from the Mason Chair Grant of CHOA awarded to S.J.K. We thank Dr. Ronald Gill (Department of
Surgery, University of Colorado-Denver) for providing KBA-1 hybridoma and anti-LFA-1 mAb; Dr.
Allan Kirk (Department of Surgery, Duke University) for helpful suggestions on the study. The authors
also would like to thank Dr. Melanie Molitor-Dart (University of Wisconsin-Madison) for performing
direct trans vivo DTH assay and Drew Roenneburg (University of Wisconsin-Madison) for performing
immunohistochemistry.
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Figure Legends
Figure 1. LFA-1 blockade prevents acute rejection of multiple minor Ag mismatched cardiac
allograft. A. Cardiac graft survival in multiple minor antigen mismatched combination. The multiple
minor Ag-mismatched combination (BALB.B into C57BL/6, minor mismatch) showed 50% of
spontaneous cardiac grafts acceptance without any manipulation (closed circle; n=24; MST >100 days)
whereas anti-LFA-1 mAb treated recipients showed no rejection (open circle; n=12; MST >100 days). B.
Beating quality of the heart grafts. The graft were monitored by daily palpation and graded from 4+
(Strongest beat) to 0 (no beat). Quality of beating from anti-LFA-1 mAb treated group (open circle) was
well preserved compared to untreated group (closed circle). C. Representative graft status in mice with
cardiac transplant by H&E, CD4, and CD8 staining. Sections showed acute rejection grade 3R of ACR at
day 10 with pronounced perivascular and multifocal interstitial lymphocytic infiltrates with foci of
myocyte damage without treatment. BALB grafts from anti-LFA-1 mAb treated recipient showed no
signs of complication at day 10 (x200). Immunohistological analysis of POD 10 allografts showed CD4
and CD8 T cell infiltration in untreated recipients while interstitial CD4 and CD8 T cell infiltration was
reduced in anti-LFA-1 mAb treated recipients. Original magnification, x200. ***P<0.001 vs. untreated
controls.
Figure 2. Endogenous alloreactive H60-specific CD8 T cells are reduced in all immune
compartments after blocking LFA-1. A.Analyses of T cell subtypes in different anatomical
compartments of C57BL/6 recipients with or without anti-LFA-1 mAb treatment at POD 10.
Mononuclear cells were isolated from spleen, blood, and heart from untreated and anti-LFA-1 mAb
treated recipients at POD10 and were examined for the expression of CD3, CD4, and CD8. Data are
plotted on the basis of CD3/CD4 or CD3/CD8 staining (as of total lymphocytes). Results are shown as
18
dot plot and each dot represents one animal. B. Phenotype of peripheral H60-specific CD8 T cell. Data
shown are expression of homing (CD11a, CD44, and CD62L) and chemokine (Cxcr3, Ccr5, and Ccr7)
receptors (open histogram) from gated H60-specific CD8 T cells. Isotype staining is shown by solid area.
C. Accumulation of H60 mH-Ag (LTFNYRNL) specific CD8 T cells in the graft. Heart sections were
incubated with FITC-conjugated CD8 antibodies plus PE-conjugated tetrameric MHC Kb-H60 peptide
complex (Kb-tetramer) and directly analyzed by CLSM without fixation. The white arrows in the insets
show the individual CD8 and H60 Tetramer staining, respectively. Merged image shows the overlay of
CD8 and H60 tetramer staining where the orange or yellow patches indicate the double-stained (H60
specific CD8 T cells) cells. Original magnification, x 40 (Inset, x200). D. Representative dot plots of
H60-specific CD8 T cells in spleen, blood and graft of untreated and anti-LFA-1 mAb treated recipients.
The percentage of H60-specific CD8 T cells was increased in all immune compartments of the untreated
animals compared to the naïve controls. However, a significant reduction of H60-specific CD8 T cells
was observed after anti-LFA-1 mAb treatment in all compartments. Gates in the figures represent the
percentage of tetramer-positive CD8 T cells within CD3+ T cells. NS>0.05, *p<0.05 vs. untreated
controls.
Figure 3. LFA-1 blockade suppresses anti-donor response including co-dominant mH-Ags-specific
CD8 T cells. A. Co-immunodominant and subdominant mH-Ags specific CD8 T cells were evaluated
from untreated and anti-LFA-1 mAb treated recipients during maximal allogeneic T cell expansion at
POD10. Three mice per each group were sacrificed 10 days after transplantation and isolated splenocytes
were stained for CD8 and epitope-specific TCRs with H4b (SGIVYIHL; H-2Kb), H7b (KAPDNRDTL; H2Db), H13b (SSVIGVWYL; H-2Db), and H28 (FILENFRPL; H-2Kb) pentamers. The dot plot represents
CD8+ gated T cells stained CD8 FITC (x-axis) and minor histocompatibility antigens (mH-Ags) specific
PE-conjugated multimers (y-axis). Gates in the figures represent the percentage of multimer-positive cells
19
within CD3+CD8+ T cells. B. Co-immunodominant H4-specific CD8 T cells showed significantly reduced
and H7-specific CD8 T cells showed strong trend of reduction after LFA-1 blockade. C. Frequency of
IFN- producing cells in naïve, untreated, and anti-LFA-1 mAb treated mH-Ags mismatched cardiac
allograft recipients as well as untreated full MHC mismatched cardiac allograft recipients were evaluated
by ELISPOT. Significantly lower frequency of IFN-producing cells compared to the fully MHC
mismatched (BALB/c to B6) yet elevated numbers of IFN- producing cells were identified in the
untreated animals compared to naïve controls. The frequency of IFN- producing cells, was back to the
pre-transplant level in anti-LFA-1 mAb treated animals. Data are expressed as the mean±SEM of three
independent experiments. *p<0.05 vs. untreated controls.
Figure 4. Effect of anti-LFA-1 mAb on development of chronic rejection. A. Representative sections
of untreated and anti-LFA-1 mAb treated grafts were stained with H&E and elastic trichrome for
evaluation of CR. Allograft was explanted 100 days after transplantation. Marked fibrointimal thickening
and fibrosis were observed from untreated recipients. B. Morphometric analysis of chronic allograft
vasculopathy. Significantly decreased neo-intimal hyperplasia, affected vessel number and fibrosis were
observed in anti-LFA-1 mAb treated recipients compared to untreated control. The following criteria was
applied for grading fibrosis; 0 (Area of fibrosis less than 5%), +1 (5-25%). +2 (51-75%), +3 (51-75%),
and +4 (>75%). Data are expressed as a bar graph of untreated (n =12) and anti-LFA-1 mAb treated
(n=12) mice. C. Immunohistological analysis of POD 100 allografts showed both CD4 and CD8 T cell
infiltration in untreated recipients while no interstitial CD4 and CD8 T cell infiltration was observed in
anti-LFA-1 mAb treated recipients. *, p<0.05 vs. untreated control
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Figure 5. Effect of anti-LFA-1 mAb on donor specific regulation. A. Reduced direct-DTH response in
anti-LFA-1 mAb treated recipients. Donor antigen (right) and an equal volume of PBS (left) were injected
into the footpad of naïve, untreated, and anti-LFA-1mAb treated C57BL/6 recipients at POD 100. DTH
responses were measured after 24 h as the change in footpad thickness (mean ± SD). Significantly
reduced net swelling (right footpad – left footpad) was observed in anti-LFA-1 mAb treated recipients.
Three different mice per group were used. B. Representative dot plot of CD4+CD25+FoxP3+ Treg cells in
spleen or after stimulation with irradiated BALB.B donor cells. Splenocytes were harvested from a naïve,
untreated (day 100) and anti-LFA-1 mAb treated (day 100) recipients and stained for CD3, CD4 and
FoxP3 mAbs. For donor stimulation, splenocytes from each mouse were co-cultured with an equal
number (4×105) of BALB.B splenocytes for 48 hours. C. Increased proliferation (by CFSE decay) was
observed in CD3+ cells from untreated (POD 10) recipients. In addition to reduced DTH response,
significantly reduced CD3+ cell proliferation was observed in anti-LFA-1 mAb treated (POD 100)
recipients. The addition of splenocytes from a-LFA-1 mAb treated recipients significantly reduced T cell
proliferation from untreated recipients. Cultured cells were stained for CD3 and analyzed by flow
cytometry. Results are representative of three to five independent experiments. Gates in the figures
represent the percentage of proliferating cells within CD3 T cells. *p<0.05 vs. untreated controls.
21
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