ICOS-Ig combined with CsA induces long term survival of cardiac allografts in mouse

आऋऑऎऊࣽईࣜऋंࣜ उँऀअࣿࣽईࣜ ࣿऋईईँःँएࣜऋंࣜऌईࣽ Journal of Medical Colleges of PLA 24 (2009) 249–258

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ICOS-Ig combined with CsA induces long term survival of cardiac allografts in mouse¯ Zhang Peng1, Wang Zhenmeng2, Qin Qin1, Tang Yi3, Wang Quanxing3, Shen Qian1* 1

Department of Clinical Diagnosis, Changhai Hospital, Second Military Medical University, Shanghai, 200433

2

Department of Anesthesiology, East Hepatobiliary Surgery Hospita, Second Military Medical University, Shanghai, 200438 3

Department of Immunology, Second Military Medical University, Shanghai, 200433 Received 9 April 2009; accepted 26 June 2009

Abstract Objective: To study the synergistic effect of ICOS-Ig combined with cyclosporine (CsA) on mouse heart transplantation and explore its therapeutic potential. Methods: ICOS-Ig fusion protein was generated by fusing the extracellular portion of human ICOS and Fc portion of human IgG. To investigate the effect of ICOS-Ig on T-cell proliferation in vitro, ICOS-Ig or IgG was added to the primary MLR cultures (BALB/c spleen T cells as responder cells and irradiated C57BL/6 spleen cells as stimulator cells). The cells responsiveness rates were detected by 3H-TdR methods. Then the T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 (donor) or C3H (third party) spleen cells as stimulator cells. To study the effect of ICOS-Ig on T-cell proliferation in vivo, CFSE-labeled C57BL/6 spleen cells were transferred to irradiated BALB/c mice. Mice were then treated with IgG, ICOS-Ig or CsA. Seventy two hours after transfer, the spleen cells of the mice were harvested for the detection of CD4+CFSE+ and CD8+CFSE+ by FACS. C57BL/6 mouse underwent transplantation of the hearts of BALB/c mouse and were then randomly divided into five equal groups: no treatment group, control IgG treated group (250 μg i.p. d2, 4, 6), ICOS-Ig treated group (250 μg i.p. d2, 4, 6), CsA treated group (10 mg/kg i.p. d0-6), ICOS-Ig combined with CsA group. The cardiac allograft survival was monitored by daily palpation. Results: In primary MLR, ICOS-Ig inhibited T-cell proliferation, (inhibition ratio 58±8.2% in 50 ȝg/ml). In secondary MLR, ICOS-Ig specifically inhibited donor spleen cells, which suggested ICOS-Ig could induce donor-specific hyporesponsiveness. In the CFSE dye assay, CD4+CFSE+ and CD8+CFSE+ in ICOS-Ig and CsA group was stronger than those in control group, which showed ICOS-Ig and CsA could inhibit

ƿ

Supported by Research Grants from the Ministry of Science and Technology of People’s Republic of China (National 863 Plan, 2002AA214091) and the National Science Foundation for Young Scholars of China (30500501).

* Corresponding author. Tel: +86 021 81873612; fax: +86 021 81873612; E-mail address: [email protected] Abbreviations: ICOS, inducible costimulatory molecule; CsA, cyclosporine; MHC, major histocompatibility complex; MLR, mixed lymphocyte reaction; CTL, cytotoxic lymphocyte reaction

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the proliferation of allo-reactive T cells in vivo. In mouse heart transplantation model, survival was significantly prolonged in animals treated with ICOS-Ig or CsA as compared with controls. Moreover, ICOS-Ig combined with CsA group had even longer engraftment (>100 d) than ICOS-Ig or CsA used alone. In histological examination, it was found that there were congestions and edemas in no treatment and IgG treated recipients, together with a lot of inflammatory cells infiltrated. Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealed milder histological changes. It was revealed in mechanical analysis that splenic T cells from recipients also exhibited depressed mixed leukocyte reactions (MLR) and cytotoxic lymphocyte reactions (CTL). Conclusion: These data suggest that ICOS-Ig combined with CsA induces a long-term survival of mouse cardiac allografts, whereas monotherapy is less effective in this regard. Thus, ICOS-Ig combined with CsA treatment may be a novel regimen to combat allograft rejection. Keywords: Inducible costimulator-Ig fusion protein; Cyclosporine; Donor-specific hyporesponsiveness; Mouse cardiac transplantation

cyclosporine (CsA) in mouse heart transplantation and

1. Introduction

explored its therapeutic potential. Many studies indicate that the blockage of CD28:B7 and CD40:CD40L costimulatory molecules is

2. Materials and methods

likely to serve as important targets for the prevention of allograft rejection and for the induction of tolerance in

2.1. Animals

animal models [1-3]. Inducible costimulatory (ICOS), with its ligand B7h, is a recently discovered

Male C57BL/6 mice (H-2b), BALB/c mice (H-2d)

costimulatory molecule of the CD28 family [4,5]. Unlike

and C3H (H-2k), 8–12 weeks of age, were purchased

some other costimulatory molecules, which are

from Shanghai Joint Venture SIPPR BK Experimental

expressed on lymphocytes constitutively, ICOS is

Animal Co. The animals were maintained under

induced only after T-cell activation, which can enhance

standard conditions and fed rodent food and water,

T-cell proliferation, cytokine production, and CD154

according to the principle of laboratory animal care and

expression and provides help for Ig production by B

the guide for the care and use of laboratory animals in

cells [6, 7]. With anti-ICOS mAb or an ICOS-Ig fusion

our institution.

-/-

protein, or transfer of grafts ICOS

recipient mice,

prolonged graft survival, although to a lesser degree

2.2. Preparation of ICOS-Ig

than CTLA4-Ig or anti-CD40L mAb therapy, chronic rejection developed with each of these treatments

The soluble form of ICOS (ICOS-Ig) was prepared

[8-11]. These findings suggest that blockade of ICOS

by constructing an mammalian expression vector

signaling can be a new therapeutic target in clinic

containing an extracellular portion of human ICOS

transplantation.

cDNA and Fc portion of human IgG. The extracellular

Because of the low efficacy of conventional

portion of human ICOS cDNA was generated from the

immunosuppressive regimens, and the consequences of

PMA(20 ng/ml)+ionomycin(1 ȝg/ml)-stimulated human

uncontrolled rejection, the costimulatory blockade is

peripheral blood leukocytes, and was amplified with

being studied in clinic as monotherapy. In this study,

primers (sense, 5’-GCGGCCCAGCCGGCCGA AATC

we investigated the effect of ICOS-Ig combined with

AATGGTTCTGCC-3’; antisense, 5’-GCTCTAGACT

Zhang Peng et al. / Journal of Medical Colleges of PLA 24 (2009) 249–258

251

TCAGCTGGCAAAG- 3’) containing Sfi I (sense) and

thymidine/well, harvested and thymidine incorporation

Xba I (antisense) restriction enzyme site sequences. The

determined.

pIGplus plasmid containing Fc portion of human IgG1 was amplified with primers (sense, 5’-TCTAGAGATCC

2.3.2. Secondary mixed leukocyte reaction (2o MLR)

CAAATCTTGTGAC-3’; antisense, 5’- GCGGC CGCT

2×106 BALB/c responder cells were co-cultured in

CATTTACCCGGAGAC AG-3’) containing Xba I

24-well plates with 2×106 irradiated (3000 rad)

(sense) and Not I (antisense) restriction enzyme site

C57BL/6 stimulator cells for 3 d in the presence of

sequences. PCR products were digested with the

ICOS-Ig or IgG1 Fc. Then, the viable cells were

appropriate restriction enzymes and cloned into a

separated from the debris by LSM and restimulated

mammalian expression vector pSecTag2. The plasmid

with the same allogeneic (C57BL/6) cells or third-party

with the correct insert was stably transfected by

(C3H) cells in 96-well round-bottom plates (for seeing

electroporation into CHO cells. The ICOS-Ig protein

primary response). No reagents were added to the

was purified from the supernatant by protein A-

second culture. Secondary response was pulsed 3 d

sepharose

later as primary response.

4FF

(Pharmacia,

Sweden)

affinity

chromatography. Recovered proteins were analyzed by SDS-PAGE in reducing and nonreductig conditions and

2.4. Effect of ICOS-Ig on T-cell proliferation in vivo

Western blotting with HRP-goat anti-human IgG1 Fc antibody (Pierce, USA). For all experiments involving

C57BL/6 spleen T cells purified by nylon wool

fusion proteins, IgG1 Fc in the absence of ICOS portion,

passage were incubated with 10 mmol/L of the tracking

a construct produced by the same cell line as ICOS-Ig,

fluorochrome CFSE (Molecular Probes, USA) [13].

was used as control.

Then, 20×106 CFSE-labeled cells were adoptively transferred to lethally irradiated (1800 rad) BALB/c

2.3. Effect of ICOS-Ig on T-cell proliferation in

recipients. Mice were subsequently allocated into four

vitro [12]

groups: (a) no treatment, (b)control IgG (0.1 mg/d), (c) ICOS-Ig (0.1 mg/d), (d) CsA (0.1 mg/d) (Novartis, o

2.3.1. Primary mixed leukocyte reaction (1 MLR) Spleens

were

major

C57BL/6 control mice also received CFSE-labeled cells.

histocompatibility complex (MHC)-mismatched mice

Three days after cell transfer, mice were sacrificed and

and prepared

by

the splenocytes were harvested and stained with either

density-gradient centrifugation using Ficoll-Hypaque

PE-anti-CD4 or PE-anti-CD8 (eBioscience, USA). By

(Pharmacia, Sweden). MLR were performed in

gating onto CD4+CFSE+ cells and CD8+CFSE+ cells,

round-bottom 96-well plates using 2×105 BALB/c

the proliferation of CD4+ and CD8+ T cells in each

responder cells and 2×105 gamma-irradiated (3000 rad)

separate generation of dividing cells could be

C57BL/6 stimulator cells in a total volume of 0.2 ml.

determined according to the CFSE profiles.

as

obtained

a single

cell

from

Switzerland). A fifth group consisting of syngeneic

suspension

ICOS-Ig or IgG1 Fc were added to the culture wells at the start of the MLR. Cultures were performed in

2.5. Cardiac transplantation procedures

triplicate and incubated at 37°C with 5% CO2 for up to 3 d. Cultures were then pulsed for 18 h with 1 PCi 3H

C57BL/6 mice were used as recipients and

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Zhang Peng et al. / Journal of Medical Colleges of PLA 24 (2009) 249–258

MHC-mismatched BALB/c mice as donors. Heterotopic

2.8. Histological examinations

cardiac transplantation to a cervical site was performed under a cuff technique as described previously [14].

The cardiac allografts were harvested 7 d after

The function of the heart was monitored daily after

transplantation. All specimens were fixed in 10%

transplantation by cervical palpation. Rejection was

buffered formalin and embedded in paraffin. Five

defined as total cessation of cardiac muscle contraction.

sections were stained with hematoxylin and eosin (HE)

To evaluate the role of ICOS-Ig and CsA in cardiac

for histological examinations.

allograft rejection, recipient mice were assigned to five experimental groups (n=15/group): (a) no treatment, (b)

2.9. Statistical analysis

treated with control IgG (250 Pg, days 0, 2, 4, 6), (c) treated with ICOS- Ig (250 Pg, days 0, 2, 4, 6), (d) CsA

All data were

expressed as mean ± SD

and

tested using the Chi square or Mann-Whitney U test. P

(10 mg/kg/d ×7 d) , and (e) ICOS- Ig and CsA combined.

value of <0.05 was considered significant. Survival was

2.6. Mixed leukocyte reactions (MLR)

evaluated by Kaplan-Meier analysis using the LogRank test.

Recipient C57BL/6 mice were sacrificed 7 d after transplantation, and the spleen T cells purified

3. Results

by nylon wool passage were used as responders. To assess the T-cell hyporesponsiveness of the recipient

3.1. Construction and expression of ICOS-Ig

mice against donor-derived cells, gamma-irradiated (3000 rad) BALB/c cells were used as stimulator

For cloning ICOS cDNA, mRNA was prepared

cells. After being cultured at 37°C for 3 d, cells were

from PMA + ionomycin-activated human peripheral

pulsed for 18 h with 1 PCi

3

H thymidine/well,

blood leukocytes. The extracellular domain (EC) of

harvested and thymidine incorporation made the

ICOS was amplified so that it terminated after Lys140,

determination.

just upstream of two cysteines near the junction with the transmembrane domain. ICOS EC domain with the

2.7. Cytotoxic lymphocyte reactions (CTL)

correct sequence was subcloned upstream of a human IgG1 Fc domain inserted into the mammalian

Cytotoxic responses were assayed by the JAM 6

expression vector pSecTag2 (Fig. 1A). Plasmid DNA

Test as previously described [15]. 5 × 10 spleen T cells

was stably transfected to CHO cells. The ICOS-Ig

from

were

protein was purified from the cell culture supernatants

stimulated for 5 d with 1×10 irradiated allogeneic

by protein A affinity chromatography. Similar to the

BALB/c spleen cells. Con A blast targets were set up

natural monovalent dimmer, ICOS-Ig seemed to be a

6

40 h prior to the CTL assay by culturing 1.5 × 10

dimeric protein about 100 kDa in molecular size, as

syngeneic and BALB/c spleen cells with ConA (1.25

judged by SDS-PAGE and Western blotting in reducing

recipient

C57BL/6

mice

(effectors)

6

3

Pg/ml), then labeling with H thymidine. Lysis of target cells was tested at various effector to target ratios.

versus nonreducing conditions (Fig. 1B and 1C).

Zhang Peng et al. / Journal of Medical Colleges of PLA 24 (2009) 249–258

253

Fig. 1. A. Structure of the ICOS-Ig fusion protein. The cDNA fragment encoding the ICOS-Ig fusion protein is diagrammed. The extracellular domain of ICOS was fused immediately

Fig. 2. A. ICOS-Ig inhibits proliferation of one-way 1° MLR.

after Lys140 to the human IgG1 Fc domain. The restriction

BALB/c spleen cells were isolated as responder cells, and

sites incorporated into the fusion gene cassette are also

gamma-irradiated C57BL/6 cells as stimulator cells. Different

indicated. B. SDS-PAGE analysis of ICOS-Ig fusion proteins

dosages of ICOS-Ig were added to primary MLR cultures

in reducing versus nonreducing conditions. Similar to the

(termed ICOS-Ig group)ēand in control group(no treatment or

natural monovalent dimmer, ICOS-Ig seems to be a dimeric

IgG). Allogeneic MLRs of cells treated with graded doses of

protein about 100 kDa in molecular size. C. Western blot analysis

ICOS-Ig or control IgG. Proliferative T cell responses can be

of ICOS-Ig fusion protein under reducing condition.

significantly inhibited by ICOS-Ig compared with control IgG. B. ICOS-Ig induces hyporesponsiveness to alloantigen

3.2. ICOS-Ig inhibits alloreactive T cell activity in

restimulation. 1° MLR cultures were incubated for 3 d in the

vitro

presence in the presence of ICOS-Ig or IgG. Then, the viable cells were separated from the debris by LSM and restimulated

To study the potential role of ICOS as a

with the same donor (C57BL/6) cells or third-party (C3H)

costimulatory molecule, we performed allogeneic

cells. 3H thymidine incorporation was determined at d3. Each

MLRs in the presence of ICOS-Ig and control IgG. To

group restimulated with third-party cells showed the maximal

monitor primary MLR, BALB/c spleen cells were

responses. IgG group restimulated with donor cells showed

isolated as responder cells, and gamma-irradiated

similar responses.The proliferation in ICOS-Ig group with

C57BL/6 cells as stimulator cells. Different dosages of

donor spleen cells was specifically inhibited in comparison

ICOS-Ig were added to primary MLR cultures (termed

with that in other groups. Error bars represent SD.

ICOS-Ig group)ēand in control group(no treatment or IgG). Incubated for 3 dēthe cells responsiveness rates

primary MLR were cultured as responder cells for

were detected by 3H thymidine methods. ICOS-Ig

secondary MLR, and irradiated C57BL/6 (donor) or

significantly inhibited T-cell proliferation, which

C3H (third-party) spleen cells as stimulator cells. The

dependedon the dosage of ICOS-Ig (inhibition ratio

proliferation of ICOS-Ig group restimulated with donor

~60% in 50 ȝg/ml) (Fig. 2A). T cells of each group in

spleen cells was specifically inhibited compared with

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Zhang Peng et al. / Journal of Medical Colleges of PLA 24 (2009) 249–258

that restimulated with third-party spleen cells, which

combined. Mice were sacrificed 3 d after cell transfer,

suggested

donor-specific

and the splenocytes were harvested and analysed by

hyporesponsiveness, that was to say, induce donor-

FACS. The results showed that CD4+ and CD8+

specific tolerance (Fig. 2B).

C57BL/6 T cells in untreated mice demonstrated strong

ICOS-Ig

could

induce

proliferative responses to BALB/c hosts. In contrast,

3.3. ICOS-Ig and/or CsA blocks allogeneic CD4+

cells transferred into syngeneic hosts underwent

and CD8+ T cell proliferation in vivo

negligible proliferation. Compared with untreated recipients, mice treated with ICOS-Ig or CsA alone had

An in vivo assay was also set up to evaluate the

a significant reduction in allo-reactive CD4+ and CD8+

impact of ICOS-Ig on allogeneic T cell proliferation.

T-cell proliferation, respectively. The most striking

CFSE-labeling C57BL/6 spleen T cells were adoptively

decay in response is illustrated in mice treated with

transferred to lethally irradiated BALB/c recipients,

ICOS-Ig and CsA combined(Fig. 3).

which were then treated by ICOS-Ig, CsA or the two

Fig. 3. ICOS-Ig and/or CsA blocks allogeneic CD4+ and CD8+ T cell proliferation in vivo. C57BL/6 spleen T cells were incubated with the tracking fluorochrome CFSE and adoptively transferred to lethally irradiated C57BL/6 (A) or BALB/c (B) recipients. Mice were subsequently allocated into five groups: (a) no treatment, (b)control IgG, (c) ICOS-Ig, (d) CsA, (e)syngeneic C57BL/6 control. 3 d after cell transfer, mice were sacrificed and the splenocytes were harvested and stained with either PE-anti-CD4 or PE-anti-CD8. By gating onto CD4+CFSE+ cells and CD8+CFSE+ cells, the proliferation of CD4+ and CD8+ T cells in each separate generation of divided cells could be determined according to the CFSE profiles.

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Zhang Peng et al. / Journal of Medical Colleges of PLA 24 (2009) 249–258

Fig. 4. A. Survival graph of cardiac allografts. Treatment with ICOS-Ig (29.5r7.7 d), CsA (21±4.5 d) significantly prolongs cardiac allograft survival as compared with untreated mice (8.5±1 d) (P<0.05). ICOS-Ig combined with CsA group induces even longer engraftment (>100 d) than ICOS-Ig or CsA used alone(P<0.01). B. Histology of murine cardiac allografts harvested 7 d after transplantation. Rejected grafts from untreated and control IgG recipients demonstrates diffuse inflammatory cell infiltration, myocyte destruction, and interstitial hemorrhage, whereas scattered inflammatory cells and relatively preserved myocytes are seen in the grafts treated with ICOS-Ig or CsA. Moreover, in ICOS-Ig combined with CsA treated group little inflammatory infiltrate are seen, and myocytes undamaged. Magnification, ‚400.

3.4. ICOS-Ig and/or CsA prolonged allograft

induced even longer engraftment (>100 d) than

survival

ICOS-Ig and CsA alone (P<0.01). (Fig. 4A). Histologic examination at 7 d after transplantation revealed that

Treatment with ICOS-Ig significantly prolonged

acute

rejection

was

significantly

suppressed

in

cardiac allograft survival (29.5r7.7 d) in comparison

allografts treated with ICOS-Ig or CsA in comparison

with that in untreated mice (8.5±1 d) (P<0.05). CsA

with that in untreated allografts. Rejected grafts from

group also similarly prolonged cardiac allograft

untreated recipients demonstrated diffuse inflammatory

survival (21±4.5 d). ICOS-Ig combined with CsA group

cell infiltration, myocyte destruction, and interstitial

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Zhang Peng et al. / Journal of Medical Colleges of PLA 24 (2009) 249–258

hemorrhage, whereas scattered inflammatory cells and relatively preserved myocytes were seen in the grafts treated with ICOS-Ig or CsA. Moreover, ICOS-Ig combined with CsA treatment almost completely suppressed lymphocytes infiltration of the grafts, and myocytes were undamaged. (Fig. 4B)

3.5. ICOS-Ig and CsA induced donor-specific hyporesponsiveness The authors performed donor-specific MLR using sensitized splenocytes from the recipients 7 d after transplantation. T-cell proliferation was significantly inhibited by ICOS-Ig or CsA compared with that in the untreated or IgG group (P<0.05). ICOS-Ig and CsA combined

synergistically

and

more

efficaciously

suppressed T-cell proliferation as compared with their respective treatment alone (P<0.05) (Fig. 5). Moreover, CTL assays were completed to evaluate anti-donor T-cell

cytotoxic

responses

demonstrating

donor-

Fig. 6. CTL responses of splenic T cells from the recipients 7

specific graft acceptance. In this assay, effector T cells

d after transplantation. A. Effector T cells from all groups has

from untreated and control IgG groups had strong CTL

similar low CTL ability to syngeneic C57BL/6 targets. B.

ability to allogeneic BALB/c target cells,

Effector T cells from untreated and control IgG groups has strong CTL ability to allogeneic BALB/c target cells, while the CTL ability of ICOS-Ig and CsA combined treated mice is significantly decreased.

while the CTL ability of ICOS-Ig and CsA treated mice was significantly decreased (Fig. 6). These results suggested that graft survival was a consequence of a long-term donor-specific hyporesponsiveness.

4. Discussion Fig. 5. The proliferation of splenic T cells from the recipients 7 d after transplantation. T-cell proliferation was significantly

Inducible costimulatory molecule (ICOS), with its

inhibited by ICOS-Ig or CsA ( P<0.05 as compared with those in

ligand B7h, is a recently discovered costimulatory

the untreated or IgG group). ICOS-Ig and CsA combined

molecule of the CD28 family [4,5]. ICOS signal

a

synergistically suppressed T-cell proliferation ( P<0.05 compared

blockade has been shown to be an effective strategy to

with their respective treatment alone).

prevent allogeneic graft rejection [8-11]. Whether

b

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Zhang Peng et al. / Journal of Medical Colleges of PLA 24 (2009) 249–258

concomitant use of ICOS blockade and conventional

response is illustrated in mice treated with combined

immunosuppressive drugs has a beneficial or adverse

ICOS-Ig and CsA.

effect on preventing rejection and promoting long-term

Then, we addressed the question as to whether the

allograft survival or transplant tolerance has not been

concomitant use of ICOS-Ig and CsA could induce the

clear so far. The present study was designed to analyse

prolongation of cardiac allograft survival. It was found

the influence of ICOS-Ig combined with CsA on

that the combined treatment resulted in long-time

cardiac allograft survival. The rational for this

survival (>100 d) of cardiac allograft, whereas ICOS-Ig

combination was based on several previous findings.

or CsA used alone was less effective. In addition to the

CsA was known to inhibit primary T-cell activation at

survival data, histological examination showed that the

the level of intracellular calcium mobilization, and thus

ICOS-Ig

downregulated

other

completely suppressed T-cell infiltration of the graft

cytokines[16]. While, ICOS signal blockade by

and cytotoxic responses in comparison with their

ICOS-Ig was effective on activated T cells because

respective treatment alone (P<0.05). We also tested that

ICOS was expressed on T cells only after stimulation,

MLR and CTL using sensitized splenocytes from the

and ICOS signaling might act synergistically with

recipients 7 d after transplantation. These results

IL-2-mediated signal transduction in T-cell activation

suggested that graft survival was a consequence of a

[17], strengthening the rationale for blockade of both

long-term donor-specific hyporesponsiveness.

expression

of

IL-2

and

and

Csa

combined

treatment

almost

pathways. Moreover, this combination was chosen

In summary, ICOS-Ig combined with CsA

based on CsA's proven efficacy clinically in transplant

treatment may be a novel post-transplantation regimen

patients [18].

to establish a donor-specific tolerant state by biological

In this study, we generated an ICOS-Ig fusion

rather than pharmacological mechanisms.

protein consisting of an extracellular portion of human ICOS and Fc portion of human IgG. We first used an

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