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The CD28 peptidemimic can induce mixed chimerism and prolong the survival of cardiac allografts
Transplant Immunology 13 (2004) 283 – 288 www.elsevier.com/locate/trim
The CD28 peptidemimic can induce mixed chimerism and prolong the survival of cardiac allografts Jin Chena,b, Qiuzao Hea,b, Huanbing Xua,b, Liping Sua,b, Jinping Zhanga,b, Sidong Xionga,b,* a
Department of Immunology and Key Laboratory of Molecular Medicine of the Ministry of Education, Fudan University, Shanghai 200032, PR China b Center for Gene Immunization and Vaccine Research, Shanghai 200032, PR China Received 14 June 2004; received in revised form 11 October 2004; accepted 12 October 2004
Abstract Costimulatory blockade with CD28 peptidemimic (CD28PM, CD28 PM was synthesized by solid phase synthetic methods) prolongs cardiac allograft survival in mice, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding B7 blockade to a chimerism inducing nonmyeloablative regimen in mice and observed a significant improvement of donor bone marrow cells (BMC) engraftment, which had been associated with mixed chimerism and long-term survival of cardiac allografts. The mixed lymphocyte reaction (MLR) and the ear pinna cardiac transplantation model were performed to evaluate the effects of CD28PM in induction of specific immune hypo-response and extension of allograft survival. The expressed rates of B7.1 and B7.2 on the C57BL/6 splenocytes were 56.25% and 20.52%, respectively. The specific hypo-response status was established after immunization with CD28PM pre-treated donor splenocytes and the average inhibition rate was only 43% compared with normal control. Subsequently, a total number of 2107 bone marrow cells per mouse were implanted to the recipients. The allogenic chimerism was obviously observed with the rate as high as 8.84% (mean) at the time point of day 14. During the first 50 days post bone marrow transfusion (BMT) the chimerism rate declined stepwise. But from 50 to 100 days, the chimerism rate sustained in a range of 3.35% to 4.6%. The results of transplantation experiments showed the survival of allgenic cardiac grafts were maintained over 100 days in recipients. Thus, donor BMC engraftment with mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Mixed chimerism approach, by the addition of CD28-B7 costimulatory blockade with CD28PM, has been shown to establish mixed chimerism and induce cardiac allograft tolerance in mice. D 2004 Elsevier B.V. All rights reserved. Keywords: Mixed chimerism; CD28; Peptidemimic; Mixed lymphocyte reaction; Cardiac allograft
1. Introduction NaRve T lymphocytes need to be activated and subsequently differentiate into effector cells to perform their immune functions. The current model of T cell activation requires two signals [1,2]. The first signal is elicited by engagement of TCR by the antigen-peptide/MHC complex. The second, costimulatory signal is provided by the interaction between accessory molecules, especially CD28, on the surface of T cells [3,4] and their ligands, such as * Corresponding author. Department of Immunology, Shanghai Medical College of Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China. Tel./fax: +86 21 54237749. E-mail address: [email protected] (S. Xiong). 0966-3274/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2004.10.002
CD80 and CD86, on APCs [5–8]. This second signal is very important for maintaining and enhancing T-cell activation [9]. When only the first signal exists, T cells would be induced to an anergy status that would result in specific T cell immune tolerance. Previously we reported a 10-amino-acid long oligopeptide (nominated CD28 peptidemimic), and synthesized it after molecular modeling. The CD28 peptidemimic could bind to B7 and lead to block the interaction of B7 and CD28. In vitro experiments show that this peptide inhibits the lymphocyte proliferation reaction [10]. Recently, we used immature DCs or resting B cells (with no or low levels of B7 expression) to induce a specific allogenic immune hypo-response in mice. Similarly, transplantation experiments also proved that this
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immune hypo-response status could specifically prolong the survival of allgenic cardiac grafts [11,12]. Yu et al. [13] exerted immature DCs to induce such hypo-response and found the hypo-response status could maintain for 7 days. During the period of hypo-response, mixed chimeras were induced after donor bone marrow cells (BMC) transfusion. In this study, CD28PM was firstly used to pretreat the irradiated C57BL/6 splenocytes in order to block the B7 molecules on them, and subsequently block the interaction of B7 and CD28. We speculated that the function of these pre-treated C57BL/6 splenocytes was similar to that of immature DCs to induce the specific hypo-response. And then donor bone marrow cells were infused to induce allogenic chimerism in recipients. The murine pinna cardiac transplantation model was then introduced for the study on the relationship between the chimerism and the survival of the grafts.
BALB/c (H-2d) new born mice (within 24 h after delivery) were purchased from experimental animal center, Shanghai Medical College, Fudan University. All animals were housed in microisolator cages, given autoclaved food, and maintained in accordance with recommendations in the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, PR China, 1998) and the guidelines of the Shanghai Medical Laboratory Animal Care and Use Committee or the Shanghai Medical College of Fudan University.
2. Materials and methods
2.4. Proliferation assay
2.1. CD28PM established and synthesis
CD28PM pretreated irradiated donor splenocytes induced a hypo-immune response status and chimerism in recipient mice. Splenocytes from donors (C57BL/6) were irradiated with 30 Gy g ray, and then pretreated with CD28PM for 90 min. After that, the cells were injected via the tail vein into recipient mice (BALB/c) at a dose of 5106/100 Al/per mouse. In 3 days, we used splenocytes from these mice as responding cells, and freshly irradiated C57BL/6 cells were used as stimulator cells to perform mixed lymphocyte proliferation reactions. The mice immunized with murine allgenic splenocytes pretreated with CD28PM were compared with mice immunized with untreated allgenic splenocytes (CD28PM ) and mice injected with physiological saline (NS) to study the effects of immune hypo-response status. Three days after immunizing mice with CD28PM pretreated allgenic splenocytes we infused freshly prepared bone marrow cells from donors (C57BL/6) at the dose of
We used the Insight II molecular modeling workstation to do molecular modeling and also used some information from related scientific papers to establish the CD28PM with the MYPPPY motif (EFMYPPPYLD). We used a solid phase method to synthesize this peptide, purified it by HPLC, and used electro spray mass-spectrum to measure its molecular weight as 1271.6. We dissolved this peptide into 1 mg/ml solution (780 umol/l), stored it at 70 8 C, and used 1640 medium to dilute it to a corresponding concentration freshly for each experiment. 2.2. Experimental animals C57BL/6(H-2Kb) mice, BALB/c (H-2Kd) mice, C3H/ HeJ (H-2Kk) mice, C57BL/6(H-2Kb) new born mice and
2.3. Flow cytometric analysis Splenocytes and peripheral blood cells in chimerism mice were directly labeled with the fluorescent antibodies (Anti-H-2Kd FITC, Anti-H-2Kb PE). The B7 molecules on C57BL/6 splenocytes were labeled with rat anti mouse CD80-FITC and CD86-FITC antibodies. These antibodies were all purchased from Pharmingen.
Fig. 1. The expression of MHC-I and B7 molecules on C57BL/6 splenocytes. Freshly prepared splenocytes derived from C57BL/6 mice were incubated for 30 min in PE-labeled antibody against H-2Kb (anti-H-2Kb–PE) or fluorescein isothiocyanate (FITC)-conjugated anti-mouse B7.1 and B7.2 antibody (anti-B7.1FITC, anti-B7.2-FITC) and the corresponding isotype control antibodies (Mouse BALB/c IgG2a, Armenian Hamster IgG2, Rat Louvain IgG2a, respectively). The splenocytes were washed twice in DPBS/FCS and analyzed by FACS assay. The H-2Kb (a), B7.1 (b) and B7.2 (c) molecules were calculated from 10,000 events collected.
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2.5. Cardiac transplantation model Split-heart neonatal cardiac grafts were transplanted into the ear pinna of mice. Allografts from donors were placed in female recipients in a pinna location as described previously [14]. Graft function was assessed daily by anatomic microscopic observation and electrocardiographic monitoring. Rejection was defined as the absence of detectable electrocardiogram (ECG) of allograft. Allografts failures or death of recipients within 48 h of surgery were considered technical failures and were excluded from the analysis. 2.6. Statistical analysis Fig. 2. Allograft hypo-responsiveness induced by allogenic splenocytes pretreated with CD28 peptidemimic. Responder cells refer to the BALB/c murine splenocytes pre-immunized with physical saline (N.S., negative control), irradiated C57BL/6 splenocytes (CD28PM , negative control) and irradiated C57BL/6 splenocytes pre-treated with CD28PM (CD28PM+, experiment group), respectively. Stimulator cells (2.5105) were mixed with responder cells (5105) and tested in mixed lymphocyte reactions (MLRs) as described in Materials and methods.
2107/100 Al/per mouse. The positive and negative controls were only injected immunosuppressor Adriblastine (Adr) at 3 mg/kg body weight via I.P. or 100 Al NS via tail vein. Two weeks later we drew blood from the recipient and used flow cytometry to monitor the H-2 phenotypes to judge the status of chimerism. We used the time points of 14, 21, 50, 80, 100 days after bone marrow infusion.
We used Stata 7.0 and SPSS 10.0 statistical software, and chose Student’s t-test, Mantel’s log rank analysis to do statistical analysis. Pb0.05 was considered significant.
3. Results 3.1. B7 and H-2Kb molecules expression on donor C57BL/6 murine splenocytes To investigate the expression of B7 and H-2Kb molecules on C57BL/6 murine splenocytes, flow cytometry was used to measure B7 expression in C57BL/6 murine splenocytes. It was found that on these splenocytes the major B7 molecule was B7.1 (CD80), 56.65%, and B7.2 (CD86) expression percentage was only 20.52%. Almost all C57BL/ 6 murine splenocytes express the murine MHC-I molecule, and the positive for H-2Kb was 99.00% (Fig. 1). 3.2. CD28PM pretreated donor splenocytes induced specific immune hypo-response in recipients The splenocytes of the BALB/c that had been immunized with the CD28PM pre-treated donor splenocytes were used
Fig. 3. Mixed chimera induced by allogenic splenocytes pre-treated with CD28PM. The irradiated splenocytes from C57BL/6(H-2Kd) mice were pretreated with CD28PM and injected into recipient BALB/c(H-2Kb) mice (5106/per mouse/100 Al). Three days later, the C57BL/6 bone marrow cells (2107/per mouse/100 Al) were infused into the recipient BALB/c mice. FACS assay was performed to analyze the chimerism state of the recipient peripheral blood mononuclear cell (PBMC) with mAbs directed to donor MHC class I (Kd; FITC, Kb; PE) 7 days after infusion. The results showed the chimerism state of one Chimera BALB/c mice on the 21, 50, 100 days after Bone Marrow Transplantation (BMT). Control group: Normal BALB/c; Normal C57BL/6; Chimeras group: Chimera BALB/c mice.
Fig. 4. Long-term allgenic chimera induced by allgenic murine splenocytes pretreated with CD28PM. The chimerism state was monitored by FACS assay on days 14, 21, 50, 80, 100 after BMT (n=6). (chimerism rate: the rate of allogenic cell of the recipient PBMC as determined by FACS assay).
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as the responder cells, and freshly prepared irradiated splenocytes from C57BL/6 were used as stimulating cells to perform mixed lymphocyte proliferation reaction. The results showed that CD28PM group had a specific immune hypo-response, its CPM was significantly inhibited compared to untreated group (NS) ( Pb0.01). The experiment was repeated three times. The average inhibition rate was 43%. However, the mice in CD28PM group had inhibition rate at 26% (Fig. 2), suggesting that CD28PM pre-treated C57BL/6 murine splenocytes can induce specific immune hypo-response in recipient BALB/c mice. 3.3. The allgenic chimera formation The recipient mice were immunized with CD28PM pretreated donor splenocytes, and were infused with freshly prepared donor (C57BL/6) bone marrow cells (2107/per mouse/100 Al) 3 days later. Two weeks later, flow cytometry was used to measure the H-2 phenotype in peripheral blood mononuclear cells (PBMC) in order to judge the status of chimerism. The results are shown in Fig. 3. The control groups are BALB/c and C57BL/6 mice. They highly expressed H-2Kd and H-2Kb, respectively. Chimeras group showed H-2Kb phenotypes for 21, 50, 100 days after BMT and chimerism rates were 6.99%, 4.90% and 3.64%, respectively, indicating allgenic chimera formation. 3.4. The duration of allgenic chimera In order to know how long the induced chimera can be maintained by CD28PM pretreated C57BL/6 murine splenocytes after BMT in BALB/c, flow cytometry was used to monitor the status of chimera on the 14, 21, 50, 80, and 100 days after BMT (n=6). It was found that chimera status could
Fig. 5. The effects of allgenic chimeras on cardiac allograft survival. Mice were treated with CD28PM pre-treated irradiated C57BL/6 splenocytes on days 3 relative to C57BL/6 BMT on day 0 (2107 BMC derived from C57BL/6(n=6)). On day 7, recipient mice were performed allogenic cardiac muscle transplantation. Control mice were treated with irradiated C57BL/6 splenocytes (CD28PM ) and Adriblastine (adr) with BMT (n=6). The survival of the allgraft was observed everyday. The allgenic mycardiac allograft survived more than 100 days.
be maintained for over 100 days. The rate of chimerism was still 3.45% 150 days after BMT (Fig. 4). 3.5. The effects on cardiac allograft survival To know whether or not the chimera status would prolong the survival of murine cardiac allograft, the donor cardiac allografts were implanted into the ear pinnas of BALB/c mice with chimerism and the survival situation of the transplanted grafts was observed. In the chimera mice, the cardiac allografts could survive more than 100 days (n=6), much longer than the control groups of Adr and CD28PM . By Log Rank analysis, v 2=30.28, Freedom m=3, Pb0.0001 (Fig. 5). Data represents two independent experiments.
4. Discussion Despite improvements in short-term results following solid organ allotransplantation, by the addition of new immunosuppressive agents, long-term results have not been significantly altered over the past decade. Therefore, induction of specific transplantation tolerance remains a major goal of transplantation immunology. One of the important strategies is induction of mixed chimerism, which has been demonstrated to be a more reliable mean of inducing transplantation tolerance in various experimental models, including nonhuman primates [15–20]. The traditional full chimerism, wherein the entire recipient hematopoietic system is destroyed and replaced by donor cells, leads to complete or near-complete donor hematopoietic reconstitution [21], however, it is very difficult to create full chimerism in large animals. Mixed chimerism, which describes a state wherein hematopoietic populations of both the recipient and donor coexist can be achieved by milder forms of treatment which do not ablate the host hematopoietic system. In addition to requiring less toxic host conditioning, mixed chimerism offers additional advantages over full chimerism, including improved immunocompetence [22,23] and reduced susceptibility to GVHD [24–26]. Earlier murine studies demonstrated that the mechanism of tolerance induction by mixed chimerism was at least in part central or clone deletion, resulting from the migration of donor cells to the thymus [27]. This requires that the recipient’s mature T cells be severely depleted so that newly maturing T cells will be clonally deleted as they repopulate the entire T-cell repertoire and provides long-lasting, durable tolerance specifically to donor tissues. Previously, we have reported the development of a nonmyeloablative conditioning regimen for induction of mixed chimerism and tolerance to cardiac allografts in MHC-mismatched mice. Our mixed chimerism conditioning regimen was based upon mouse studies from our department which revealed that mixed chimerism and tolerance can be induced by immature DCs without complete T-cell depletion.
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Among the multiple T-cell activation pathways, CD28/ B7 costimulatory signals have been demonstrated to be critical not only for T-cell–antigen presenting cell (APC) interactions, but also for T–B-cell interactions. Blocking of CD28/B7 interactions by CD28 monoclonal antibody (mAb),CTLA4-Ig [28–30], anti CD40 mAb [31] in animal models represents a powerful strategy for preventing graft rejection [32]. Because of immature DCs with no or low B7 molecule on their surface, we used immature DCs to precondition the recipient in order to induce damned T cell falling into anergy statue, which caused recipients to develop a specific immune hypo-response to the donor, thus, generated a time-window for donor bone marrow transplantation. Finally, a mixed allgenic chimera was successfully induced in recipient. However, it was painstaking to prepare the immature DCs. Recently, it was reported some small peptides or non-peptides compound can act as antagonists for the interaction between proteins [33]. The interaction of CD28 and B7 is restricted at a conserved complementarity determining regions (CDRs) [34]. In this study, our results showed that on the surface of C57BL/6 murine splenocytes, 50.65% of B7 was B7.1, and B7.2(CD86) was only 20.52%. We had previously used the Insight II molecule modeling system to screen the CD28 Peptidemimic among a series of candidate peptides contained the MYPPPY motif and finally confirmed the sequence of CD28PM was EFMYPPPYLD. Molecular modeling and in vitro experiments also confirm that CD28PM can bind B7.1 strongly to block the interaction of B7.1 and CD28. When CD28PM pretreated donor splenocytes are infused into recipients, the second signal (CD28/B7) for immune rejection response in the recipient is pre-blocked. It causes inactivation of related responsive T cells. The in vitro study showed that its immunoreactivity was only 43% of normal mice. This provides a tolerance time-window for bone marrow transfusion (BMT). If we do BMT during this period, we can successfully induce a mixed chimera that can last more than 100 days. Allgenic pinna cardiac transplantation also proved that the grafts could be long-term accepted in recipient (last more than 100 days). In the present study, the addition of a short course of CD28PM pre-treated donor splenocytes to our standard regimen induced mixed chimerism more consistently, and significantly enhanced the duration of chimerism achieved. This improved consistency of mixed chimerism has been associated with a lower incidence of acute rejection and comparable long-term survival of the cardiac allograft. Others have also reported that induction of stable mixed chimerism in mice is possible, without cytoreductive conditioning, if low-dose donor bone marrow cells and splenocytes plus adenovirus encoding for CTLA4Ig gene promote stable mixed chimerism and long-term survival of rat cardiac allografts [35]. Our results have demonstrated, for the first time, that CD28PM blockade can also promote hematopoietic cell engraftment and enhance chimerism in
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mice. This study not only proves that blocking B7-CD28/ CTLA4 can induce a specific immune hypo-response in the recipient, but also it can be used to induce mixed chimera formation. This method is inexpensive, simple and may be an important method for anti-allografts rejection and prolongation of the grafts survival.
Acknowledgments The authors thank Bing Qiao, Hong Xiaowu, Yang Xiuli, Li Ang, Deng Fugang for helpful discussions. We thank Ren Xuefang and Xu Ling for assistance with animal experiments. Funding: This work supported by the National Basic Research Program of China (2001CB510005), National Natural Science Funds of China (no. 39830340), Graduate Innovation Foundation of Fudan University.
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The CD28 peptidemimic can induce mixed chimerism and prolong the survival of cardiac allografts Jin Chena,b, Qiuzao Hea,b, Huanbing Xua,b, Liping Sua,b, Jinping Zhanga,b, Sidong Xionga,b,* a
Department of Immunology and Key Laboratory of Molecular Medicine of the Ministry of Education, Fudan University, Shanghai 200032, PR China b Center for Gene Immunization and Vaccine Research, Shanghai 200032, PR China Received 14 June 2004; received in revised form 11 October 2004; accepted 12 October 2004
Abstract Costimulatory blockade with CD28 peptidemimic (CD28PM, CD28 PM was synthesized by solid phase synthetic methods) prolongs cardiac allograft survival in mice, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding B7 blockade to a chimerism inducing nonmyeloablative regimen in mice and observed a significant improvement of donor bone marrow cells (BMC) engraftment, which had been associated with mixed chimerism and long-term survival of cardiac allografts. The mixed lymphocyte reaction (MLR) and the ear pinna cardiac transplantation model were performed to evaluate the effects of CD28PM in induction of specific immune hypo-response and extension of allograft survival. The expressed rates of B7.1 and B7.2 on the C57BL/6 splenocytes were 56.25% and 20.52%, respectively. The specific hypo-response status was established after immunization with CD28PM pre-treated donor splenocytes and the average inhibition rate was only 43% compared with normal control. Subsequently, a total number of 2107 bone marrow cells per mouse were implanted to the recipients. The allogenic chimerism was obviously observed with the rate as high as 8.84% (mean) at the time point of day 14. During the first 50 days post bone marrow transfusion (BMT) the chimerism rate declined stepwise. But from 50 to 100 days, the chimerism rate sustained in a range of 3.35% to 4.6%. The results of transplantation experiments showed the survival of allgenic cardiac grafts were maintained over 100 days in recipients. Thus, donor BMC engraftment with mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Mixed chimerism approach, by the addition of CD28-B7 costimulatory blockade with CD28PM, has been shown to establish mixed chimerism and induce cardiac allograft tolerance in mice. D 2004 Elsevier B.V. All rights reserved. Keywords: Mixed chimerism; CD28; Peptidemimic; Mixed lymphocyte reaction; Cardiac allograft
1. Introduction NaRve T lymphocytes need to be activated and subsequently differentiate into effector cells to perform their immune functions. The current model of T cell activation requires two signals [1,2]. The first signal is elicited by engagement of TCR by the antigen-peptide/MHC complex. The second, costimulatory signal is provided by the interaction between accessory molecules, especially CD28, on the surface of T cells [3,4] and their ligands, such as * Corresponding author. Department of Immunology, Shanghai Medical College of Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China. Tel./fax: +86 21 54237749. E-mail address: [email protected] (S. Xiong). 0966-3274/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2004.10.002
CD80 and CD86, on APCs [5–8]. This second signal is very important for maintaining and enhancing T-cell activation [9]. When only the first signal exists, T cells would be induced to an anergy status that would result in specific T cell immune tolerance. Previously we reported a 10-amino-acid long oligopeptide (nominated CD28 peptidemimic), and synthesized it after molecular modeling. The CD28 peptidemimic could bind to B7 and lead to block the interaction of B7 and CD28. In vitro experiments show that this peptide inhibits the lymphocyte proliferation reaction [10]. Recently, we used immature DCs or resting B cells (with no or low levels of B7 expression) to induce a specific allogenic immune hypo-response in mice. Similarly, transplantation experiments also proved that this
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immune hypo-response status could specifically prolong the survival of allgenic cardiac grafts [11,12]. Yu et al. [13] exerted immature DCs to induce such hypo-response and found the hypo-response status could maintain for 7 days. During the period of hypo-response, mixed chimeras were induced after donor bone marrow cells (BMC) transfusion. In this study, CD28PM was firstly used to pretreat the irradiated C57BL/6 splenocytes in order to block the B7 molecules on them, and subsequently block the interaction of B7 and CD28. We speculated that the function of these pre-treated C57BL/6 splenocytes was similar to that of immature DCs to induce the specific hypo-response. And then donor bone marrow cells were infused to induce allogenic chimerism in recipients. The murine pinna cardiac transplantation model was then introduced for the study on the relationship between the chimerism and the survival of the grafts.
BALB/c (H-2d) new born mice (within 24 h after delivery) were purchased from experimental animal center, Shanghai Medical College, Fudan University. All animals were housed in microisolator cages, given autoclaved food, and maintained in accordance with recommendations in the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, PR China, 1998) and the guidelines of the Shanghai Medical Laboratory Animal Care and Use Committee or the Shanghai Medical College of Fudan University.
2. Materials and methods
2.4. Proliferation assay
2.1. CD28PM established and synthesis
CD28PM pretreated irradiated donor splenocytes induced a hypo-immune response status and chimerism in recipient mice. Splenocytes from donors (C57BL/6) were irradiated with 30 Gy g ray, and then pretreated with CD28PM for 90 min. After that, the cells were injected via the tail vein into recipient mice (BALB/c) at a dose of 5106/100 Al/per mouse. In 3 days, we used splenocytes from these mice as responding cells, and freshly irradiated C57BL/6 cells were used as stimulator cells to perform mixed lymphocyte proliferation reactions. The mice immunized with murine allgenic splenocytes pretreated with CD28PM were compared with mice immunized with untreated allgenic splenocytes (CD28PM ) and mice injected with physiological saline (NS) to study the effects of immune hypo-response status. Three days after immunizing mice with CD28PM pretreated allgenic splenocytes we infused freshly prepared bone marrow cells from donors (C57BL/6) at the dose of
We used the Insight II molecular modeling workstation to do molecular modeling and also used some information from related scientific papers to establish the CD28PM with the MYPPPY motif (EFMYPPPYLD). We used a solid phase method to synthesize this peptide, purified it by HPLC, and used electro spray mass-spectrum to measure its molecular weight as 1271.6. We dissolved this peptide into 1 mg/ml solution (780 umol/l), stored it at 70 8 C, and used 1640 medium to dilute it to a corresponding concentration freshly for each experiment. 2.2. Experimental animals C57BL/6(H-2Kb) mice, BALB/c (H-2Kd) mice, C3H/ HeJ (H-2Kk) mice, C57BL/6(H-2Kb) new born mice and
2.3. Flow cytometric analysis Splenocytes and peripheral blood cells in chimerism mice were directly labeled with the fluorescent antibodies (Anti-H-2Kd FITC, Anti-H-2Kb PE). The B7 molecules on C57BL/6 splenocytes were labeled with rat anti mouse CD80-FITC and CD86-FITC antibodies. These antibodies were all purchased from Pharmingen.
Fig. 1. The expression of MHC-I and B7 molecules on C57BL/6 splenocytes. Freshly prepared splenocytes derived from C57BL/6 mice were incubated for 30 min in PE-labeled antibody against H-2Kb (anti-H-2Kb–PE) or fluorescein isothiocyanate (FITC)-conjugated anti-mouse B7.1 and B7.2 antibody (anti-B7.1FITC, anti-B7.2-FITC) and the corresponding isotype control antibodies (Mouse BALB/c IgG2a, Armenian Hamster IgG2, Rat Louvain IgG2a, respectively). The splenocytes were washed twice in DPBS/FCS and analyzed by FACS assay. The H-2Kb (a), B7.1 (b) and B7.2 (c) molecules were calculated from 10,000 events collected.
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2.5. Cardiac transplantation model Split-heart neonatal cardiac grafts were transplanted into the ear pinna of mice. Allografts from donors were placed in female recipients in a pinna location as described previously [14]. Graft function was assessed daily by anatomic microscopic observation and electrocardiographic monitoring. Rejection was defined as the absence of detectable electrocardiogram (ECG) of allograft. Allografts failures or death of recipients within 48 h of surgery were considered technical failures and were excluded from the analysis. 2.6. Statistical analysis Fig. 2. Allograft hypo-responsiveness induced by allogenic splenocytes pretreated with CD28 peptidemimic. Responder cells refer to the BALB/c murine splenocytes pre-immunized with physical saline (N.S., negative control), irradiated C57BL/6 splenocytes (CD28PM , negative control) and irradiated C57BL/6 splenocytes pre-treated with CD28PM (CD28PM+, experiment group), respectively. Stimulator cells (2.5105) were mixed with responder cells (5105) and tested in mixed lymphocyte reactions (MLRs) as described in Materials and methods.
2107/100 Al/per mouse. The positive and negative controls were only injected immunosuppressor Adriblastine (Adr) at 3 mg/kg body weight via I.P. or 100 Al NS via tail vein. Two weeks later we drew blood from the recipient and used flow cytometry to monitor the H-2 phenotypes to judge the status of chimerism. We used the time points of 14, 21, 50, 80, 100 days after bone marrow infusion.
We used Stata 7.0 and SPSS 10.0 statistical software, and chose Student’s t-test, Mantel’s log rank analysis to do statistical analysis. Pb0.05 was considered significant.
3. Results 3.1. B7 and H-2Kb molecules expression on donor C57BL/6 murine splenocytes To investigate the expression of B7 and H-2Kb molecules on C57BL/6 murine splenocytes, flow cytometry was used to measure B7 expression in C57BL/6 murine splenocytes. It was found that on these splenocytes the major B7 molecule was B7.1 (CD80), 56.65%, and B7.2 (CD86) expression percentage was only 20.52%. Almost all C57BL/ 6 murine splenocytes express the murine MHC-I molecule, and the positive for H-2Kb was 99.00% (Fig. 1). 3.2. CD28PM pretreated donor splenocytes induced specific immune hypo-response in recipients The splenocytes of the BALB/c that had been immunized with the CD28PM pre-treated donor splenocytes were used
Fig. 3. Mixed chimera induced by allogenic splenocytes pre-treated with CD28PM. The irradiated splenocytes from C57BL/6(H-2Kd) mice were pretreated with CD28PM and injected into recipient BALB/c(H-2Kb) mice (5106/per mouse/100 Al). Three days later, the C57BL/6 bone marrow cells (2107/per mouse/100 Al) were infused into the recipient BALB/c mice. FACS assay was performed to analyze the chimerism state of the recipient peripheral blood mononuclear cell (PBMC) with mAbs directed to donor MHC class I (Kd; FITC, Kb; PE) 7 days after infusion. The results showed the chimerism state of one Chimera BALB/c mice on the 21, 50, 100 days after Bone Marrow Transplantation (BMT). Control group: Normal BALB/c; Normal C57BL/6; Chimeras group: Chimera BALB/c mice.
Fig. 4. Long-term allgenic chimera induced by allgenic murine splenocytes pretreated with CD28PM. The chimerism state was monitored by FACS assay on days 14, 21, 50, 80, 100 after BMT (n=6). (chimerism rate: the rate of allogenic cell of the recipient PBMC as determined by FACS assay).
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as the responder cells, and freshly prepared irradiated splenocytes from C57BL/6 were used as stimulating cells to perform mixed lymphocyte proliferation reaction. The results showed that CD28PM group had a specific immune hypo-response, its CPM was significantly inhibited compared to untreated group (NS) ( Pb0.01). The experiment was repeated three times. The average inhibition rate was 43%. However, the mice in CD28PM group had inhibition rate at 26% (Fig. 2), suggesting that CD28PM pre-treated C57BL/6 murine splenocytes can induce specific immune hypo-response in recipient BALB/c mice. 3.3. The allgenic chimera formation The recipient mice were immunized with CD28PM pretreated donor splenocytes, and were infused with freshly prepared donor (C57BL/6) bone marrow cells (2107/per mouse/100 Al) 3 days later. Two weeks later, flow cytometry was used to measure the H-2 phenotype in peripheral blood mononuclear cells (PBMC) in order to judge the status of chimerism. The results are shown in Fig. 3. The control groups are BALB/c and C57BL/6 mice. They highly expressed H-2Kd and H-2Kb, respectively. Chimeras group showed H-2Kb phenotypes for 21, 50, 100 days after BMT and chimerism rates were 6.99%, 4.90% and 3.64%, respectively, indicating allgenic chimera formation. 3.4. The duration of allgenic chimera In order to know how long the induced chimera can be maintained by CD28PM pretreated C57BL/6 murine splenocytes after BMT in BALB/c, flow cytometry was used to monitor the status of chimera on the 14, 21, 50, 80, and 100 days after BMT (n=6). It was found that chimera status could
Fig. 5. The effects of allgenic chimeras on cardiac allograft survival. Mice were treated with CD28PM pre-treated irradiated C57BL/6 splenocytes on days 3 relative to C57BL/6 BMT on day 0 (2107 BMC derived from C57BL/6(n=6)). On day 7, recipient mice were performed allogenic cardiac muscle transplantation. Control mice were treated with irradiated C57BL/6 splenocytes (CD28PM ) and Adriblastine (adr) with BMT (n=6). The survival of the allgraft was observed everyday. The allgenic mycardiac allograft survived more than 100 days.
be maintained for over 100 days. The rate of chimerism was still 3.45% 150 days after BMT (Fig. 4). 3.5. The effects on cardiac allograft survival To know whether or not the chimera status would prolong the survival of murine cardiac allograft, the donor cardiac allografts were implanted into the ear pinnas of BALB/c mice with chimerism and the survival situation of the transplanted grafts was observed. In the chimera mice, the cardiac allografts could survive more than 100 days (n=6), much longer than the control groups of Adr and CD28PM . By Log Rank analysis, v 2=30.28, Freedom m=3, Pb0.0001 (Fig. 5). Data represents two independent experiments.
4. Discussion Despite improvements in short-term results following solid organ allotransplantation, by the addition of new immunosuppressive agents, long-term results have not been significantly altered over the past decade. Therefore, induction of specific transplantation tolerance remains a major goal of transplantation immunology. One of the important strategies is induction of mixed chimerism, which has been demonstrated to be a more reliable mean of inducing transplantation tolerance in various experimental models, including nonhuman primates [15–20]. The traditional full chimerism, wherein the entire recipient hematopoietic system is destroyed and replaced by donor cells, leads to complete or near-complete donor hematopoietic reconstitution [21], however, it is very difficult to create full chimerism in large animals. Mixed chimerism, which describes a state wherein hematopoietic populations of both the recipient and donor coexist can be achieved by milder forms of treatment which do not ablate the host hematopoietic system. In addition to requiring less toxic host conditioning, mixed chimerism offers additional advantages over full chimerism, including improved immunocompetence [22,23] and reduced susceptibility to GVHD [24–26]. Earlier murine studies demonstrated that the mechanism of tolerance induction by mixed chimerism was at least in part central or clone deletion, resulting from the migration of donor cells to the thymus [27]. This requires that the recipient’s mature T cells be severely depleted so that newly maturing T cells will be clonally deleted as they repopulate the entire T-cell repertoire and provides long-lasting, durable tolerance specifically to donor tissues. Previously, we have reported the development of a nonmyeloablative conditioning regimen for induction of mixed chimerism and tolerance to cardiac allografts in MHC-mismatched mice. Our mixed chimerism conditioning regimen was based upon mouse studies from our department which revealed that mixed chimerism and tolerance can be induced by immature DCs without complete T-cell depletion.
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Among the multiple T-cell activation pathways, CD28/ B7 costimulatory signals have been demonstrated to be critical not only for T-cell–antigen presenting cell (APC) interactions, but also for T–B-cell interactions. Blocking of CD28/B7 interactions by CD28 monoclonal antibody (mAb),CTLA4-Ig [28–30], anti CD40 mAb [31] in animal models represents a powerful strategy for preventing graft rejection [32]. Because of immature DCs with no or low B7 molecule on their surface, we used immature DCs to precondition the recipient in order to induce damned T cell falling into anergy statue, which caused recipients to develop a specific immune hypo-response to the donor, thus, generated a time-window for donor bone marrow transplantation. Finally, a mixed allgenic chimera was successfully induced in recipient. However, it was painstaking to prepare the immature DCs. Recently, it was reported some small peptides or non-peptides compound can act as antagonists for the interaction between proteins [33]. The interaction of CD28 and B7 is restricted at a conserved complementarity determining regions (CDRs) [34]. In this study, our results showed that on the surface of C57BL/6 murine splenocytes, 50.65% of B7 was B7.1, and B7.2(CD86) was only 20.52%. We had previously used the Insight II molecule modeling system to screen the CD28 Peptidemimic among a series of candidate peptides contained the MYPPPY motif and finally confirmed the sequence of CD28PM was EFMYPPPYLD. Molecular modeling and in vitro experiments also confirm that CD28PM can bind B7.1 strongly to block the interaction of B7.1 and CD28. When CD28PM pretreated donor splenocytes are infused into recipients, the second signal (CD28/B7) for immune rejection response in the recipient is pre-blocked. It causes inactivation of related responsive T cells. The in vitro study showed that its immunoreactivity was only 43% of normal mice. This provides a tolerance time-window for bone marrow transfusion (BMT). If we do BMT during this period, we can successfully induce a mixed chimera that can last more than 100 days. Allgenic pinna cardiac transplantation also proved that the grafts could be long-term accepted in recipient (last more than 100 days). In the present study, the addition of a short course of CD28PM pre-treated donor splenocytes to our standard regimen induced mixed chimerism more consistently, and significantly enhanced the duration of chimerism achieved. This improved consistency of mixed chimerism has been associated with a lower incidence of acute rejection and comparable long-term survival of the cardiac allograft. Others have also reported that induction of stable mixed chimerism in mice is possible, without cytoreductive conditioning, if low-dose donor bone marrow cells and splenocytes plus adenovirus encoding for CTLA4Ig gene promote stable mixed chimerism and long-term survival of rat cardiac allografts [35]. Our results have demonstrated, for the first time, that CD28PM blockade can also promote hematopoietic cell engraftment and enhance chimerism in
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mice. This study not only proves that blocking B7-CD28/ CTLA4 can induce a specific immune hypo-response in the recipient, but also it can be used to induce mixed chimera formation. This method is inexpensive, simple and may be an important method for anti-allografts rejection and prolongation of the grafts survival.
Acknowledgments The authors thank Bing Qiao, Hong Xiaowu, Yang Xiuli, Li Ang, Deng Fugang for helpful discussions. We thank Ren Xuefang and Xu Ling for assistance with animal experiments. Funding: This work supported by the National Basic Research Program of China (2001CB510005), National Natural Science Funds of China (no. 39830340), Graduate Innovation Foundation of Fudan University.
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