文档库

最新最全的文档下载
当前位置:文档库 > Yihong Huang-Relieving GVHD by mPEG-SPA and anti-OX40L

Yihong Huang-Relieving GVHD by mPEG-SPA and anti-OX40L

ORIGINAL ARTICLE

Ef?cacy of pretreatment of allografts with methoxypolyethylene glycol-succinimidyl-propionic acid ester in combination with an anti-OX40L monoclonal antibody in relieving graft-versus-host disease in mice

Yihong Huang ?Saran Feng ?Renxian Tang ?

Bing Du ?Kailin Xu ?Xiuying Pan

Received:21July 2009/Revised:30August 2010/Accepted:27September 2010/Published online:17October 2010óThe Japanese Society of Hematology 2010

Abstract We designed to determine whether pretreat-ment of allografts with methoxypolyethylene glycol-suc-cinimidyl-propionic acid ester (mPEG-SPA)and an anti-OX40L monoclonal antibody (McAb)can relieve acute graft-versus-host disease in allogeneic bone marrow transplantation recipients.Responder splenocytes from C57BL/6donor mice were incubated with stimulator splenocytes from BALB/c recipient mice for 7days in the presence or absence of anti-OX40L McAb followed by mPEG-SPA modi?cation.Donor BM cells plus mixed culture T cells were then transplanted into myeloablatively irradiated BALB/c mice.The signs of GVHD were less evident in mice of groups B (mPEG-SPA modi?cation group),C (anti-OX40L McAb pretreated group)and D (dual-treated group),with average survival durations all longer than those in group A (non-treated BMT group)(P \0.05).The survival rates on day 60post-BMT in groups B,C and D were 50,41.7and 66.7%,respectively.After BMT,serum IL-4and IL-10levels elevated in groups B,C (P \0.05)and even more signi?cantly increased in group D (P \0.01),while serum IFN-gamma levels decreased in these three groups (P \0.01).In conclusion,the combination of mPEG-SPA and anti-OX40L McAb can block T cell-activated antigens,co-stimulatory pathways and induce the immune shift of Th cells toward Th2cells;their effects in ameliorating GVHD are synergistic.

Keywords Methoxypolyethylene glycol áChemical modi?cation áAnti-OX40L monoclonal antibody áImmune tolerance áGraft-versus-host disease

1Introduction

These days,allogeneic bone marrow transplantation (allo-BMT)has been accepted as the most effective treatment for a variety of hematological malignancies.However,acute graft-versus-host disease (aGVHD)which is mediated by alloantigen-activated donor T cells is still a major obstacle to long-term success of allo-BMT [1,2].Severe aGVHD has high levels of morbidity and mortality.Most thera-peutic approaches designed to alleviate aGVHD have focused on the development of immunosuppressive agents and the ex vivo removal of donor T cells from the marrow.Unfortunately,T cell depletion has been associated with increased rates of engraftment failure,sustained immuno-suppression and leukemia relapse.Therefore,a desirable therapy will be needed to reduce the incidence and severity of aGVHD and improve the survival rate of recipients.Strategies for the reliable induction of a robust,permanent state of immunological tolerance have the potential to improve dramatically the prospects for allograft recipi-ents [3].

The chemical modi?er methoxypolyethylene glycol (mPEG)is a type of amphoteric compound without immunogenicity and is used to modify various proteins covalently and to prepare versatile blood types.It has no toxicity or immunogenicity and does not accumulate in the human body or affect the biological functions of its substrates [4,5].Additionally,mPEG and its derivative,mPEG-succinimidyl-propionic acid ester (mPEG-SPA)are two commonly used chemical modi?ers that have been

Y.Huang (&)áS.Feng áR.Tang áB.Du áK.Xu áX.Pan Department of Hematology,

Af?liated Hospital of Xuzhou Medical College,Xuzhou 221002,China

e-mail:hxr1583@http://www.wendangku.net/doc/063a62400722192e4536f690.html;hxr1583@http://www.wendangku.net/doc/063a62400722192e4536f690.html

Int J Hematol (2010)92:609–616DOI 10.1007/s12185-010-0701-y

approved by the FDA for clinical use[4].By mPEGyla-tion,mPEG is covalently attached to the protein antigens on the surface of lymphocytes,forming a?exible hydro-philic shell that masks antigenic sites and effectively prevents any speci?c immune response[6].Meanwhile, OX40and OX40-ligand(OX40L)are another important pair of co-stimulatory signaling molecules except for the CD28/B7pathway.OX40/OX40L mainly function at a late stage of T cell activation to prolong the survival and proliferation of activated T lymphocytes through syner-gistically boosting the responses to CD4?T lymphocytes mediated by CD28/B7[7].Therefore,blocking the OX40/ OX40L co-stimulatory signaling pathway can inhibit the activation of T lymphocytes and induce immune tolerance [8].The activation of donor T lymphocytes is a key step in GVHD development[9,10].Blocking T cell activation antigens and inhibiting the activation of speci?c co-stimulatory receptors may be an ideal strategy for induc-ing immune tolerance and thus preventing and relieving GVHD.

This study used an animal model to show that in vitro mPEG-SPA modi?cation and incubation with anti-OX40L monoclonal antibody(McAb)can induce effective immune tolerance of donor splenocytes and acts synergistically to relieve and inhibit aGVHD.Collectively,this study pro-vides experimental evidence for the prophylaxis of aGVHD after allo-BMT.

2Materials and methods

2.1Materials

2.1.1Experimental animals

Male C57BL/6(H-2b)mice were chosen as donors and female BALB/c(H-2d)as recipients.All mice were of clean grade,aged8–12weeks and weighed18–20g.All mice were purchased from the Experimental Animal Cen-ter of Xuzhou Medical College.Animals were handled according to Animal Ethics Committee guidelines.

2.1.2Reagents

mPEG-SPA was purchased from the Beijing Kaizheng Biotech Development Co.,Ltd and rat anti-mouse OX40L antibody from the eBioscience Corporation.Red blood cell lysates,PE-conjugated mouse anti-CD4and anti-CD8 monoclonal antibodies and FITC-conjugated mouse anti-CD3and anti-2D b monoclonal antibodies were purchased from BD Biosciences(USA).IL-4,IL-10and IFN-gamma ELISA kits were purchased from Shenzhen Jingmei Bio-tech Co.,Ltd.Calf serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co.,Ltd and lymphocyte separation medium from the Tianjin Haoyang Biological Manufacture Co.,Ltd.

3Methods

3.1Preparation of donor splenocytes

3.1.1Incubation of donor and recipient splenocytes

with anti-OX40L McAb

Splenocytes from donor and recipient mice were col-lected under sterile conditions and used as response and stimulatory cells.Splenocytes from BALB/c recipient mice were treated with mitomycin at a?nal concentra-tion of25l g/mL at37°C in a cell incubator containing 5%CO2for30min,washed twice to remove residual mitomycin and used as stimulatory cells.Response and stimulatory cells(49106/well)were seeded on a 12-well plate and cultured in2mL RPMI1640in the presence or absence of10l g/mL anti-OX40L McAb at 37°C in a cell incubator containing5%CO2.Cell culture media were changed every other day and antibodies were added at a concentration of10l g/mL after the?rst change of the media.The cells were collected at the7th day,washed with PBS twice and adjusted to39107 cells/mL with RPMI1640.Cell viability was analyzed by trypan staining,indicating cell survival rates of [95%.

3.1.2Modi?cation of donor splenocytes by mPEG-SPA

Two hours before transplantation,anti-OX40L pretreated or untreated donor splenocytes(49106/well)were incu-bated with15mg/mL mPEG-SPA for60min at pH8.0 and4°C,washed with PBS twice and adjusted to39107 cells/mL.Cell survival rates of[95%were demonstrated by trypan staining.Cells were then double stained with FITC-conjugated mouse anti-CD3McAb and PE-conju-gated anti-CD4or anti-CD8McAbs.Cell surface CD3, CD4and CD8expressions were analyzed with?ow cytometry to determine the antigen masking effect of mPEG-SPA.

3.1.3Preparation of bone marrow cell suspension

from donor mice

Donor C57BL/6mice were killed by cervical dislocation. Bone marrow cells were collected from the tibia and femur in sterile conditions,washed twice with PBS,and adjusted to a concentration of39107cells/mL.Trypan blue staining indicated cell survival rates of[95%.

610Y.Huang et al.

3.2Replication of GVHD model in mice

In the experiments,female BALB/c mice were used as recipient mice.Five days before transplantation,bedding material was replaced and drinking water changed to cool boiled water containing250mg/L erythrocin and320mg/L gentamycin.BALB/c mice were myeloablatively irradiated with8.5Gy total body irradiation(TBI)by60Co c-ray (35.5–50.0cGy/min)in a vertical position4h before transplantation and subsequently transplanted with bone marrow cells and splenocytes from C57BL/6mice at a ratio of1:1.

3.3Animal grouping and transplantation

Irradiated BALB/c recipient mice were randomized into four groups:group A(allo-BMT control group,n=12) mice were transfused with69106donor bone marrow cells and69106untreated splenocytes;group B(mPEG-SPA modi?cation group,n=12)mice were transfused with69106bone marrow cells and69106mPEG-SPA modi?ed splenocytes;group C(anti-OX40L McAb pre-treated group,n=12)mice were transfused with69106 bone marrow cells and69106anti-OX40L McAb pre-treated splenocytes;group D(mPEG-SPA and anti-OX40L McAb dual-treated group,n=12)mice were transfused with69106bone marrow cells and69106mPEG-SPA modi?ed and anti-OX40L McAb pretreated splenocytes. As well,?ve mice were not transfused with any donor cells.The TBI group was used to ensure that the recipient mice were all myeloablatively irradiated.

Equal volumes of bone marrow cells and pretreated splenocytes suspensions from C57BL/6donor mice were gently mixed.A total of0.4mL cell mixture(129106 cells)was transfused into each BALB/c recipient mouse from the tail vein on the day of the TBI pretreatment.

3.4Clinical assessment of GVHD

After BMT,mice were scored for clinical evidence of GVHD.General conditions such as body weight,eating and mental state of recipient mice were observed regularly (twice a week in the?rst2weeks and once a week after-ward)to examine the emergence of aGVHD signs, including hunching,piloerection,diarrhea,weight loss and depression.The survival duration of each recipient mouse was recorded and the survival rate of each group calculated accordingly.Survival of longer than60days with normal body weight,diet and activity was considered long-term survival.The degree of systemic GVHD was assessed by a standard scoring system that included?ve clinical param-eters,i.e.,weight loss,posture(hunching),activity,fur texture and skin integrity.At the time of the analyses,mice from coded cages were evaluated and graded from0to2 for each criterion.A clinical index was subsequently generated by summation of the?ve criterion scores (maximum index=10)[11].Pad skin,liver and intestinal tissues from mice with GVHD-related death and mice killed on day30post-BMT were subjected to pathological examinations and classi?ed according to the GVHD path-ological grading system[12,13].Hematopoietic recovery was regularly evaluated by the proportion of peripheral white blood cells(WBCs)in blood smear examinations. WBCs\0.59109/L and[1.09109/L were considered to indicate transplant failure and hematopoietic recovery, respectively.Deaths occurring within2weeks after BMT caused by infection,bleeding and other non-GVHD-related hematopoietic suppressive effects were de?ned as BMT-related deaths.

3.5Flow cytometry analysis of T lymphocyte subsets

after BMT

Peripheral blood from the recipient mice(40l L)was collected by tail vein puncture on days1,5,10,15,20,25 and30post-BMT,mixed with2l L heparin and divided into two equal parts.The blood was dual-?uorescently stained with1l L FITC-conjugated anti-CD25antibody and1l L PE-conjugated mouse anti-CD4or anti-CD8 antibody and subjected to?ow cytometry analysis for CD4-and CD8-positive T lymphocytes.The ratio of CD4?T/ CD8?T lymphocytes was calculated accordingly.

3.6ELISA detection of the peripheral blood cytokine

Peripheral blood from the recipient mice(1.5mL)was collected via the fossa orbitalis vein on days1,5,10,15, 20,25and30post-BMT and centrifuged for serum,which was preserved at-80°C prior to use.The levels of IFN-gamma,IL-4and IL-10in peripheral blood serum were detected with ELISA kits according to the instructions by the manufacturer.

3.7Detection of donor chimerism

On day60post-BMT,two BALB/c recipient mice were randomly chosen from each group and killed and bone marrow cell suspensions routinely prepared.Bone marrow cells(19106/L)were washed twice with PBS,incubated with PE-conjugated anti-H-2b McAb for20min in the dark and treated with hemolysin for another5min in the dark to dissolve erythrocytes.Cells were subsequently washed twice with PBS and suspended in0.5mL PBS.Chimerism was analyzed with a?ow cytometer using CellQuest soft-ware to determine the proportion of donor lymphocytes (H-2b?uorescence intensity)in the recipient system.

Relieving GVHD by mPEG-SPA and anti-OX40L611

3.8Statistical analysis

Estimated parameters of quantity measures are presented as mean±standard deviation(x±s)and processed with GraphPad Prism4.0software.Differences were statisti-cally analyzed with one-way ANOVA.Inter-group com-parisons were performed with Tukey’s q test.Survival curves were calculated by the Kaplan–Meier method and compared using the log-rank test.Tests providing proba-bilities P\0.05were considered statistically signi?cant. 4Results

4.1mPEG-SPA effectively masked antigens

on the surfaces of splenocytes

Donor splenocytes were pretreated with15mg/mL of mPEG-SPA for chemical modi?cation[14].The levels of cell surface CD3,CD4and CD8were detected by antibody

staining and?ow cytometry analysis.The detectable level of each receptor was signi?cantly reduced,showing a masking rate higher than88%(Table1).Cell survival rates of[95% were demonstrated by trypan staining.These results indi-cated that15mg/mL of mPEG-SPA effectively masked cell surface antigens without affecting splenocyte viability. 4.2Clinical signs and pathological changes of aGVHD

in recipient mice after BMT

The?ve mice in the TBI group all died within9days after irradiation.Their WBCs were lower than0.59109/L, indicating myeloablative irradiation.Recipient mice in group A were observed with poor general conditions after transplantation,included exhaustion,anorexia,weight loss, hunching,progressive emaciation,rough and shineless hair coat and a severe decline in activity between days8and13 post-BMT.Their WBCs were higher than19109/L before death.Four(1/3)mice in group A exhibited dermal ulcers,diarrhea and other clinical signs of aGVHD.The clinical aGVHD score for mice in group A was 7.17±0.72(maximum score of10).Pathological exam-inations of skin,liver and intestine tissues showed grades 3–4GVHD damage.

Nevertheless,aside from temporary weight loss,only some of the mice in groups B,C and D developed clinical signs of aGVHD;overall these signs were mild,such as hunching,hair erection and diarrhea between days9and15 post-BMT.The clinical aGVHD scores in mice of groups B,C and D were2.50±1.00,2.67±1.16and1.33±0.49,respectively,signi?cantly lower than that of group A (P\0.05).The clinical aGVHD score of mice of group D was also statistically lower than those of groups B and C (P\0.05).Pathological examinations suggested grades 1–3aGVHD damage in dying mice and grades1–2(if any) aGVHD damage in mice with long-term survival.

4.3In vitro pretreatments of allo-grafts improved

post-BMT survival of recipient mice

All mice in group A died of aGVHD between days7and 17post-BMT.The average survival durations were sig-ni?cantly higher in groups B(36.17±24.93days),C (32±24.78days)and D(44.33±23.19days)than in group A(12.1±5.5days,P\0.05).The average survival duration of mice in group D was the longest among all the recipient mice.The survival rates of mice60days after BMT were also signi?cantly elevated in groups B(50%, 6/12),C(41.7%,5/12)and D(66.7%,7/12)compared to group A(0%,0/12).Mice in group D had the highest survival rate(P\0.05);the survival rates in groups B and C were not statistically different(Fig.1).

Table1Proportions of CD3?, CD4?and CD8?T lymphocytes before and after mPEG-SPA modi?cation(n=6,%)

CD3?T lymphocytes CD4?T lymphocytes CD8?T lymphocytes

Before modi?cation52.27±2.7035.42±1.3127.84±1.85

After modi?cation 6.61±0.85 3.52±0.35 2.82±0.25 Covering rate0.88±0.020.90±0.010.89±

Yihong Huang-Relieving GVHD by mPEG-SPA and anti-OX40L

0.01

Fig.1Post-BMT survival curves of mice in different transplantation

groups.Each experiment group versus control group,P\0.05;

mPEG-SPA group versus combined group,P\0.05;anti-OX40L

McAb group versus combined group P\0.05

612Y.Huang et al.

4.4Peripheral T lymphocyte subsets in recipient mice Flow cytometry analysis of peripheral T lymphocyte sub-sets indicated that the peripheral CD4?T proportions and CD4?/CD8?ratios on days 5,10and 15post-BMT were statistically lower in groups B (P \0.05)and C (P \0.05)and even lower in mice of group D (P \0.01)than in group A.The proportions of CD8?T lymphocytes in mice of groups B and D peaked on day 10post-BMT and were also evidently higher than in group A (P \0.05for both).The proportions of CD4?T and CD8?T lymphocytes and CD4?/CD8?ratios were similar between mice in groups B and D (P [0.05,Fig.2).

4.5Serum cytokine levels in recipient mice

Time-dependent changes in serum IFN-gamma,IL-4and IL-10in each group of recipient mice are shown in Fig.3.After transplantation,serum IFN-gamma levels in mice of group A were gradually elevated and peaked between days 10and 15post-BMT.Serum IFN-gamma levels were decreased in mice of groups B,C and D and reached the lowest levels on day 10post-BMT.Serum IFN-gamma levels were the most signi?cantly downregulated in mice of group D (P \0.01).IFN-gamma levels were not statisti-cally different between mice of groups B and C (P [0.05).Serum IL-4and IL-10levels were slightly decreased in group A and markedly increased in groups B,C and D (P \0.05)and peaked between days 10and 15post-BMT in these groups.The levels of IL-4and IL-10were the most evidently up-regulated in group D (P \0.01);no statistical difference was observed between IL-4or IL-10levels of groups B and C (P [0.05).

4.6Donor chimerism after myeloablative BMT

The proportions of H-2b -positive cells in recipient mice were analyzed with ?ow cytometry on day 60post-BMT.These results indicated that the chimerism rate was 95–100%in all surviving recipient mice,with complete donor-type

Yihong Huang-Relieving GVHD by mPEG-SPA and anti-OX40L

implantation.

Fig.2Post-BMT changes in T lymphocyte subsets.a Proportions of CD4?T lymphocytes after BMT in different transplantation groups.*

P \0.05versus control group;**P \0.01versus control group;#

P \0.05versus combined group.b Proportions of CD8?T lympho-cytes after BMT in different transplantation groups.*P \0.05versus control group;**P \0.001versus control group;#P \0.05versus mPEG-SPA group and combined group.c Post-BMT CD4?/CD8?ratios in different transplantation groups.*P \0.05versus control group;**P \0.001versus control group;#P \0.05versus combined group;D P \0.05versus anti-OX40L McAb group

c Relieving GVHD by mPEG-SPA an

d anti-OX40L

613

5Discussion

Graft-versus-host disease is caused by administration of donor T cells into genetically disparate recipients.In?am-matory cytokine release has been implicated as the primary mediator of acute GVHD,where the activation of T cells is one step in the process of pathophysiology [15,16].Since it has been well recognized that T cells activation plays a key role in initiating GVHD,searching for substances that can mask the receptors and antigens on the cell surfaces of donor T lymphocytes is a promising orientation to inhibit the activation and proliferation of donor T cells and the secretion of cytokines.It has been reported that chemical modi?cations can effectively mask cell surface antigens and prevent the activation of T lymphocytes.Previous studies have shown that with an increase in the concen-tration of mPEGylation,the surface antigen expression in lymphocytes becomes dramatically down-regulated,indi-cating a negative correlation between these two variables.The shielding effects of mPEG on surface antigen expression did not alter with culture time,indicating mPEG can clearly and in a sustainable manner mask surface antigens in lymphocytes [14,17].In this study,15mg/mL mPEG-SPA was used to modify donor T lymphocytes and masked cell surface CD3by 88%.Transfusion of donor T lymphocytes modi?ed by mPEG-SPA in vitro signi?cantly attenuated the clinical manifestations and pathological grade of aGVHD and evidently improved the overall sur-vival of recipient mice.These results strongly suggest that mPEG-SPA modi?cation to grafts can prevent and relieve GVHD.We hypothesize that T lymphocyte activation might be reduced as cell surface antigens,such as CD3,are masked preventing signals from host HLA alloantigens from effectively being transferred to intracellular signaling molecules.Provided this condition exists,the ability of donor T lymphocytes to proliferate and respond is greatly impaired,which suppresses immune rejection and inhibits the occurrence of GVHD.Nevertheless,cell surface anti-gens cannot be entirely blocked and the activation of T lymphocytes can be regulated by a number of factors,so that donor T lymphocytes can still be activated,but to a lesser extent and mice transfused with modi?ed T lym-phocytes can still exhibit some mortality.To improve prevention of GVHD,other strategies and methods must be applied to induce further the immune tolerance of donor T lymphocytes by inhibiting their responses to host HLA alloantigens.

OX40L (CD252)is the ligand for OX40(CD134)and is expressed on cells such as DC2s (a subtype of dendritic cells),enabling ampli?cation of T cell differentiation [18,19].Anti-OX40L McAb can inhibit the activation of

Yihong Huang-Relieving GVHD by mPEG-SPA and anti-OX40L

T

Fig.3Post-BMT serum IL-4,IL-10and IFN-gamma levels in different transplantation groups

614Y.Huang et al.

lymphocytes and suppresses the immune responses of donor lymphocytes by blocking the binding of OX40L to OX40on the surface of donor T lymphocytes and interrupt the OX40-OX40L signaling pathway,thus relieving GVHD[20,21].Two experimental strategies have been used to study the impact of the OX40/OX40L co-stimulatory signaling pathway in the generation of GVHD:intraperi-toneal injection of anti-OX40L McAb and an OX40gene-knockout mouse http://www.wendangku.net/doc/063a62400722192e4536f690.htmling these two methods to block the OX40/OX40L co-stimulatory signaling pathway in vivo partly alleviates the generation of GVHD[22,23]. Nevertheless,in vivo application of anti-OX40L McAb would require large amounts of antibodies be highly toxic and the establishment of a gene-knockout mouse model is technically challenging.To overcome these obstacles and reduce the side effects of in vivo antibody application,we pre-incubated donor T lymphocytes with an anti-OX40L McAb in vitro and mixed them with donor bone marrow cells before transfusion to investigate whether such treat-ment could also relieve GVHD in mice.CD4?T lympho-cytes play an important role in the pathological process of initiating and amplifying GVHD[24].Consistent with a number of previous studies[10,19],the proportions of donor CD8?T and especially CD4?T lymphocytes decreased after anti-OX40L McAb-treated splenocytes were transfused into the recipient mice.The proliferation pro?le of T lymphocytes after transplantation implied that both activation and proliferation of T lymphocytes became signi?cantly inhibited immediately after BMT and gradu-ally entered a relatively quiescent state,likely representing the maintenance phase of immune tolerance.In addition, the low level of CD4?T lymphocytes was probably asso-ciated with the decrease of memory T cells induced by blocking the OX40/OX40L signaling pathway,as well as the inhibition of T cells activation which played a role in regulating secondary immune responses.CD4?T lympho-cytes are divided into Th1and Th2cells based on the cytokines produced and active proteins.In murine models, donor CD4?Th1cells preferentially secrete type I cyto-kines(IL-2and IFN-gamma)and induce signi?cant GVHD,whereas donor Th2cells that primarily secrete type II cytokines(IL-4,IL-5,IL-10and IL-13)reduce GVHD and down-regulate GVHD initiated by Th1/Tc1cells[25]. Our study suggests that blocking OX40L with the anti-OX40L McAb before transfusion could signi?cantly down-regulate the production of the Thl-type cytokine IFN-gamma and up-regulate the secretion of Th2-type cytokines IL-4and IL-10in recipient mice.These results indicate that in addition to its association with‘‘replication incompetence’’of T lymphocytes,the immune tolerance induced by OX40/OX40L signaling blockage is also correlated with an increased differentiation of Th0cells to Th2cells.This study indicates that blocking OX40/OX40L with anti-OX40L McAb could clearly relieve GVHD in mice.Since GVHD is regulated by multiple co-stimulatory molecules and other signaling pathways,involving cyto-kines and lymphocytes[26,27],it cannot be completely inhibited with this single approach.

In the present study,mPEG-SPA was used in combination with an anti-OX40L McAb to pre-treat allogeneic graft cells before allo-BMT.We conclude that mice in the dual-treated group had an obviously lower incidence and severity of post-BMT GVHD than those in the anti-OX40L McAb pretreated and mPEG-SPA modi?cation groups.In the dual-treated group,about66.7%of recipient mice had long-term survival and stable chimerism.With respect to the secretion of cyto-kines,the combined use of mPEG-SPA and anti-OX40L McAb reduced the secretion of the Th1-type cytokine IFN-gamma,suggesting that its combined use simultaneously acted on CD8?T and perhaps more strongly on CD4?T cells and exerted its effects on the late stage of CD4?T cell acti-vation.The combined use also increased the production of the Th2-type cytokines IL-4and IL-10,which further demon-strates that the combined use of mPEG-SPA and anti-OX40L McAb could synergistically promote the transition of Th to Th2and facilitate the immune tolerance of donor CD4?T cells toward host HLA alloantigens.In short,this combination can synergistically reduce the incidence of aGVHD in mice, relieve the clinical symptoms of aGVHD and prolong the survival of recipient mice.Collectively,the results of this study provide experimental evidence for the prevention of aGVHD after allo-BMT.

Although we achieved surprising progress in our study in terms of antigen and co-stimulatory regulation by combining the use of mPEG-SPA and anti-OX40L McAb, further studies should be conducted to discuss unresolved questions,such as how mPEG negatively affects other tissues and cells.Would antigen masking cells be exposed to cellular surface again?Is it possible to block entirely the co-stimulatory regulation due to its multiple transduction pathways?As well the question,whether the graft-versus-leukemia effect would be weakened should be addressed. To resolve these questions,we will extensively and intensively conduct more studies using big animals for the purpose of clinical application.

Acknowledgments HYH and FSR contributed equally to this manuscript.This work was supported by the Natural Science Foun-dation of Jiangsu Province for Universities(07KJD320224)and the Research Projects of Xuzhou(XM07C067).

Con?ict of interest The authors declare that they have no con?ict of interest.

Relieving GVHD by mPEG-SPA and anti-OX40L615

References

1.Schmitz N,Eapen M,Horowitz MM,Zhang MJ,Klein JP,Rizzo

JD,et al.Long-term outcome of patients given transplants of mobilized blood or bone marrow:a report from the International Bone Marrow Transplant Registry and the European Group for blood and marrow transplantation.Blood.2006;108:4288–90. 2.Eapen M,Horowitz MM,Klein JP,Champlin RE,Loberiza FR

Jr,Ringden O,et al.Higher mortality after allogeneic peripheral-blood transplantation compared with bone marrow in children and adolescents:the Histocompatibility and Alternate Stem Cell Source Working Committee of the International Bone Marrow Transplant Registry.J Clin Oncol.2004;22:4872–80.

3.Newell KA,Larsen CP.Transplantation tolerance.Semin

Nephrol.2007;27:487–97.

4.Baker DP,Lin EY,Lin K,Pellegrini M,Petter RC,Chen LL,

et al.N-terminally PEGylated human interferon-beta-1a with improved pharmacokinetic properties and in vivo ef?cacy in a melanoma angiogenesis model.Bioconjug Chem.2006;17: 179–88.

5.Gundersen SI,Palmer AF.Conjugation of methoxypolyethylene

glycol to the surface of bovine red blood cells.Biotechnol Bio-eng.2007;96:1199–210.

6.Chen AM,Scott MD.Immunocamou?age:prevention of trans-

fusion-induced graft-versus-host disease via polymer grafting of donor cells.J Biomed Mater Res A.2003;67:626–36.

7.Hochweller K,Sweenie CH,Anderton SM.Circumventing tol-

erance at the T cell or the antigen-presenting cell surface:anti-bodies that ligate CD40and OX40have different effects.Eur J Immunol.2006;36:389–96.

8.Wang G,Feng Y,Hao J,Li A,Gao X,Xie S.Induction of

xenogeneic islet transplantation tolerance by simultaneously blocking CD28-B7and OX40-OX40L co-stimulatory pathways.

Sci China C Life Sci.2005;48:515–22.

9.Iwasaki T.Recent advances in the treatment of graft-versus-host

disease.Clin Med Res.2004;2:243–52.

10.Kim MY,Bekiaris V,McConnell FM,Gaspal FM,Raykundalia

C,Lane PJ.OX40signals during priming on dendritic cells inhibit CD4T cell proliferation:IL-4switches off OX40signals enabling rapid proliferation of Th2effectors.J Immunol.

2005;174:1433–7.

11.Cooke KR,Kobzik L,Martin TR,Brewer J,Delmonte J Jr,

Crawford JM,et al.An experimental model of idiopathic pneu-monia syndrome after bone marrow transplantation:I.The roles of minor H antigens and endotoxin.Blood.1996;88:3230–9. 12.Asai O,Longo DL,Tian ZG,Hornung RL,Taub DD,Ruscetti

FW,et al.Suppression of graft-versus-host disease and ampli?-cation of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation.J Clin Invest.

1998;101:1835–42.

13.Sun K,Wilkins DE,Anver MR,Sayers TJ,Panoskaltsis-Mortari

A,Blazar BR,et al.Differential effects of proteasome inhibition by bortezomib on murine acute graft-versus-host disease

(GVHD):delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity.Blood.

2005;106:3293–9.

14.Yang G,Tang SQ,Wang JW,Liu Y,Liu LZ,Feng C.Ex vivo

chemical modi?cation of graft cells by methoxy polyethylene glycol alleviates graft versus host disease after haploidentical stem cell transplantation in mice.Zhongguo Shi Yan Xue Ye Xue Za Zhi.2007;15:1240–2.

15.Sun Y,Tawara I,Toubai T,Reddy P.Pathophysiology of acute

graft-versus-host disease:recent advances.Transl Res.

2007;150:197–214.

16.Reddy P,Ferrara JL.Immunobiology of acute graft-versus-host

disease.Blood Rev.2003;17:187–94.

17.Oshima M,Atassi MZ.T cells of mice treated with mPEG-my-

asthenogenic peptide conjugate are involved in protection against EAMG by stimulating lower pathogenic antibody responses.

Autoimmunity.2000;32:45–55.

18.Maizels RM,Yazdanbakhsh M.Immune regulation by helminth

parasites:cellular and molecular mechanisms.Nat Rev Immunol.

2003;3:733–44.

19.Serghides L,Bukczynski J,Wen T,Wang C,Routy JP,Boulassel

MR,et al.Evaluation of OX40ligand as a costimulator of human antiviral memory CD8T cell responses:comparison with B7.1 and4-1BBL.J Immunol.2005;175:6368–77.

20.Taylor PA,Panoskaltsis-Mortari A,Freeman GJ,Sharpe AH,

Noelle RJ,Rudensky AY,et al.Targeting of inducible costimu-lator(ICOS)expressed on alloreactive T cells down-regulates graft-versus-host disease(GVHD)and facilitates engraftment of allogeneic bone marrow(BM).Blood.2005;105:3372–80.

21.Mendel I,Shevach EM.Activated T cells express the OX40

ligand:requirements for induction and costimulatory function.

Immunology.2006;117:196–204.

22.Blazar BR,Sharpe AH,Chen AI,Panoskaltsis-Mortari A,Lees C,

Akiba H,et al.Ligation of OX40(CD134)regulates graft-versus-host disease(GVHD)and graft rejection in allogeneic bone marrow transplant recipients.Blood.2003;101:3741–8.

23.Tsukada N,Akiba H,Kobata T,Aizawa Y,Yagita H,Okumura

K.Blockade of CD134(OX40)-CD134L interaction ameliorates lethal acute graft-versus-host disease in a murine model of allo-geneic bone marrow transplantation.Blood.2000;95:2434–9. 24.Edinger M,Hoffmann P,Ermann J,Drago K,Fathman CG,

Strober S,et al.CD4?CD25?regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation.Nat Med.2003;9:1144–50.

25.Fowler DH.Shared biology of GVHD and GVT effects:potential

methods of separation.Crit Rev Oncol Hematol.2006;57:225–44.

26.Ruggeri L,Mancusi A,Burchielli E,Aversa F,Martelli MF,

Velardi A.Natural killer cell alloreactivity and haplo-identical hematopoietic transplantation.Cytotherapy.2006;8:554–8. 27.Remberger M,Jaksch M,Uzunel M,Mattsson J.Serum levels of

cytokines correlate to donor chimerism and acute graft-vs.-host disease after haematopoietic stem cell transplantation.Eur J Haematol.2003;70:384–91.

616Y.Huang et al.