Shuiping Jiang

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Activated CD1d-restricted natural killer T cells secrete Il-2: innate help for CD4+CD25+ regulatory T cells?

CD4+CD25+ and CD1d‐restricted natural killer T (NKT) cells are thymus‐derived self‐reactive regulatory T cells that play a key role in the control of pathological immune responses. Little is known about functional cooperation between innate regulatory NKT cells and adaptive CD4+CD25+ regulatory cells. Here we show that human CD4+Vα24+Vβ11+ (CD4+ NKT) cells isolated from peripheral blood by flow cytometric cell sorting secrete substantial amounts of IL‐2 after stimulation with dendritic cells (DC) and α‐Galactosylceramide. When cocultured with CD4+CD25+ cells, CD4+ NKT cells promoted moderate proliferation of CD4+CD25+ cells. The proliferation of CD4+CD25+ T cells was due to soluble IL‐2 produced by activated CD4+ NKT cells. The expanded CD4+CD25+ cells remained anergic and retained their potent suppressive properties. These findings indicate that unlike conventional CD4+ and CD8+ T cells, which are susceptible to CD4+CD25+ regulatory cell suppression, NKT cells promote CD4+CD25+ regulatory cell proliferation. These data raise the possibility that NKT cells can function as helper cells to CD4+CD25+ regulatory T cells, thereby providing a link between the two naturally occurring populations of regulatory T cells.

Generation and expansion of human CD4+CD25+ regulatory T cells with indirect allospecificity : Potential reagents to promote donor-specific transplantation tolerance

Background. Harnessing naturally arising CD4+CD25+ regulatory T cells (Tregs) for potential adoptive cell therapy is hampered by their innate autoreactivity and their limited number. Methods. CD4+CD25+ Tregs were purified from peripheral blood of human leukocyte antigen (HLA) DR1*0101+A2− individuals, and stimulated with autologous monocyte-derived dendritic cells (DCs). Results. Here we show that CD4+CD25+ Tregs specific for an HLA A2 (103–120) peptide can be selected from the peripheral blood CD4+CD25+ T cell population of a healthy individual and detected using a tetramer comprised of HLA DRB1*0101 and the A2 peptide. The selected cells can be expanded substantially (i.e., a 1600-fold increase over a two-week period) by T-cell receptor (TCR) stimulation and high-doses of interleukin-2 (IL-2). The CD4+CD25+ Tregs with indirect allospecificity for the A2 peptide showed more potent antigen-specific suppression than polyclonal CD4+CD25+ Tregs. Conclusions. These data may pave the way for clinical studies using CD4+CD25+ Tregs with indirect allospecificity as therapeutic reagents for the induction of donor-specific transplantation tolerance.

Indefinite mouse heart allograft survival in recipient treated with CD4(+)CD25(+) regulatory T cells with indirect allospecificity and short term immunosuppression.

CD4(+)CD25(+) regulatory T cells (Tregs) play a crucial role in controlling immune responses. It is an appealing strategy to harness Tregs for adoptive cell therapy to induce tolerance to allografts. Several approaches have been developed to expand antigen-specific Tregs. Despite the large body of experimental data from murine studies demonstrating the great potential of these cells for clinical application, Treg adoptive transfer therapy was used in immunodeficient animals or in strain combinations with limited histiocompatibility. The aim of this study was to investigate whether Treg lines can protect from allograft rejection in a fully MHC-mismatched strain combination and whether the presence of Tregs with indirect allospecificity offered an advantage compared to self-reactive Tregs. Treg lines with self-specificity or with indirect allospecificity were generated by stimulating BL/6 CD4(+)CD25(+) T cells with autologous immature DCs either unpulsed or pulsed with K(d) peptide. The Treg lines were injected into recipient mice in combination with temporary depletion of CD8(+) T cells and a short course of Rapamycin. The data demonstrate that Treg lines with indirect allospecificity can be generated and most importantly they can induce indefinite survival of BALB/c hearts transplanted into BL/6 recipients when combined with short term immunosuppression. However, the Treg lines with self-specificity were only slightly less effective. The data presented in this study demonstrate the potential of ex vivo expanded Treg lines for adoptive cell therapy to promote transplantation tolerance.

CD4+CD25+ regulatory T-cell therapy for allergy, autoimmune disease and transplant rejection.

Naturally occurring CD4+CD25+ regulatory T cells (Tregs) play a critical role in the control of periphery tolerance to self-antigens. Interestingly, they also control immune responses to allergens and transplant antigens. Recent studies in animal models have shown that adoptive transfer of CD4+CD25+ Tregs can prevent or even cure allergic and autoimmune diseases, and appear to induce transplantation tolerance. Thus, adoptive cell therapy using patient-specific CD4+CD25+ Tregs has been emerged as individualized medicine for the treatment of inflammatory disease including allergy, autoimmune disease and transplant rejection. Furthermore, strategies to activate and expand antigen-specific CD4+CD25+ Tregs in vivo using pharmacological agents may represent a novel avenue for drug development.

CD4+CD25+ regulatory T cell therapy for the induction of donor-specific clinical transplantation tolerance

Naturally occurring CD4+CD25+ regulatory T cells (Tregs) have been shown to play a key role in the control of autoimmunity. Interestingly, they are also capable of mediating transplantation tolerance and they can have indirect allospecificity for donor antigens. An increasing body of evidence in experimental studies has indicated that adoptive transfer of in vitro expanded CD4+CD25+ Tregs with indirect antidonor allospecificity can induce long-term donor-specific transplantation tolerance. Thus, adoptive cell therapy using patient-specific CD4+CD25+ Tregs as individualised medicine to promote clinical transplantation tolerance is promising.

Regulatory T cells: from bench to bedside.

Regulatory T cells (Tregs) are subsets of T cells that are specifically dedicated to controlling immune responses. It has been established that Tregs play a key role in regulating autoimmune disease, allergy, cancer, infectious disease, and in the induction of transplantation tolerance. The latest progress in the development, function, mechanism of action, and homeostasis of regulatory T cells, and their translation to the clinic were presented at the International Conference on Regulatory T Cells and Clinical Application in Human Diseases in Beijing on 25-27 October 2008 (China Tregs 2008). In this Special Issue of International Immunopharmacology, several papers submitted to the China Tregs 2008 will highlight some of the recent advances in the biology of regulatory T cells and current strategies to translate regulatory T cells into the clinic.

CD4 + CD25 + regulatory T-cell therapy

Naturally occurring CD4+CD25+ regulatory T cells (Tregs) expressing forkhead transcript factor box P3 have been established as a key regulator for the control of autoimmunity. A growing body of experiments in animal models has provided compelling evidence that the adoptive transfer of CD4+CD25+ Tregs can cure many autoimmune and allergic diseases, and appear to induce donor-specific transplantation tolerance. Thus, customer-tailored CD4+CD25+ Tregs have emerged as potential reagents for adoptive cell therapy for individualized medicine. This review discusses recent advances towards that goal.

CD4+CD25+ Regulatory T Cell Therapy for the Induction of Clinical Transplantation Tolerance

The pursuit of transplantation tolerance is still in progress some 53 years after Medawar and colleagues’ first description. It has been established beyond doubt that regulatory T cells can confer donor-specific tolerance in mouse models of transplantation. However, this is crucially dependent on the strain combination, the organ transplanted and most importantly, the ratio of Tregs to alloreactive effector T cells. The ex-vivo expansion of Tregs is one solution to increase the number of alloantigen specific cells capable of suppressing the alloresponse. This technique has been used to demonstrate long term graft survival in mouse models, where ex-vivo expanded, alloantigen specific T cells are shown to preferentially migrate to, and proliferate in, the graft and draining lymph node. When such models are selected to test the role of the different allorecognition pathways for Treg induced graft survival, it appears that only a modest direct pathway alloresponse is sufficient to abrogate tolerance in immunocompetent mice. This remains the case when Tregs are expanded with both direct and indirect pathway allospecificity. Therefore, in human transplantation it is likely that depletion of the majority of direct pathway alloreactive T cells will be required to tip the balance in favour of regulation. Ex-vivo expansion of alloantigen specific, indirect pathway human Tregs, which can cross regulate the residual direct pathway has been established. Rapid expansion of these cells is possible, while they retain antigen specificity, suppressive properties and favourable homing markers. Furthermore, considerable progress has been made in the last few years to define which immunosuppressive drugs favour the expansion and function of Tregs. It is proposed that a trial of Treg therapy in combination with depletion of alloreactive T cells and short term immunosuppression is on the near horizon for human transplantation.

Generation of CD4+CD25+ regulatory T cells with indirect alloresponses by T cell receptor gene transfer: A novel therapeutic agent for the induction of transplantation tolerance.

1 A Novel siRNA-Containing Solution Protecting Donor Organs in Heart Transplantation. Xiufen Zheng,1 Dameng Lian,1 Mu Li,1 Xusheng Zhang,1 Mahdieh Khoshniat,3 Hongtao Sun,1 Weihua Liu,1 Jifu Jiang,1 Dong Chen,1 Costin Vladau,1 Motohiko Suzuki,1 James C. Lacefi eld,3 Bertha Carcia,1,2 Anthony M. Jevnikar,1,2,3 Wei-Ping Min.1,2,3 1Department of Surgery, Pathology, and Microbiology & Immunology, University of Western Ontario, London, ON, Canada; 2Multi-Organ Transplant Program, London Health Sciences Centre, London, ON, Canada; 3Robarts Research Institute, London, ON, Canada. Background: Ischemia-reperfusion (I/R) injury occurs in organ transplantation. We hypothesize that siRNA can effectively suppress infl ammatory, apoptosis and complement genes which contribute to I/R injury. This study was designed to develop a novel siRNA-containing preservation solution, which offers an alternative for protection of donor organs. Methods: Multiple siRNAs that specifi cally target TNFα, Fas, and C3 genes were prepared using our newly-developed bio-synthesis method. Heart grafts from BALB/c mice were preserved in UW solution (control) or siRNA-containing UW solution (siRNA solution) at 4°C for 48 hrs, and subsequently transplanted into syngeneic BALB/c mice. Cardiac functions were quantitatively assessed by High Frequency Ultrasound. The I/R injury was assessed by immunohistochemistry. Results: After 48 hrs preservation in the siRNA solution, the expressions of TNFα, Fas, and C3 genes in the grafts were inhibited at mRNA and protein levels detected by qPCR and immunoblot. Using siRNA solution preserved organ as donor, the graft survival was signifi cantly prolonged in heart transplantation. While siRNA solution-treated heart grafts retained strong heartbeat up to the end point of observation (>70 days), the control grafts lost function within 7 days. In addition, an improved cardiac function was observed in the graft preserved in siRNA solution. Grafts treated by siRNA solution displayed normal function, whereas grafts preserved in control solution showed dysfunction as detected by ultrasound scanning. The protection of graft by siRNA solution is associated with prevention of I/R injury. siRNA solution-treated organs exhibited almost normal histological structures, less neutrophil and lymphocyte infi ltration as compared with control solution-treated organs. Furthermore, siRNA solution prevented apoptosis and necrosis of cardiac tissues. Conclusions: This is the fi rst demonstration of a novel siRNA solution that can prevent cardiac I/R injury, protect cardiac function, and prolong graft survival in heart transplantation, implying a signifi cant and potential application in clinic. Abstract# 2 The Long-Term Evaluation of HLA-Mismatched Combined Kidney and Bone Marrow Transplant Recipients after Complete Withdrawal of Immunosuppression. Tatsuo Kawai,1 David H. Sachs,2 Thomas Spitzer,3 Nina Tolkoff-Rubin,1 Susan Saidman,4 Winfred Williams,1 Bimalangshu Dey,3 Nelson Goes,1 Dicken Ko,1 Waichi Wong,1 Robert B. Colvin,4 Megan Sykes,2 A. Benedict Cosimi.1 1Transplantation Unit, Massachusetts General Hospital, Boston, MA; 2Trasnplant Biology Research Center, MGH, Charlestown, MA; 3Bone Marrow Transplant Unit, MGH, Boston, MA; 4Pathology, MGH, Boston, MA; 5this Study was Performed in Partnership with Immune Tolerance Network. BACKGROUND: Induction of allograft tolerance has been the ultimate goal in organ transplantation. METHODS: Five subjects with ESRD received combined kidney and bone marrow transplantation (CKBMT) from HLA mismatched donors. The preparative regimen consisted of cyclophosphamide, thymic irradiation, anti-CD2 mAb and a 9-14 months course of a calcineurin inhibitor. Two subjects also received two doses of antiCD20 mAb prior to transplant. RESULTS: All recipients developed transient mixed chimerism without GVHD.Complete withdrawal of immunosuppressive medication was achieved in 4/5 recipients with current immunosuppression-free survival exceeding 3.2, 2 The Long-Term Evaluation of HLA-Mismatched Combined Kidney and Bone Marrow Transplant Recipients after Complete Withdrawal of Immunosuppression. Tatsuo Kawai,1 David H. Sachs,2 Thomas Spitzer,3 Nina Tolkoff-Rubin,1 Susan Saidman,4 Winfred Williams,1 Bimalangshu Dey,3 Nelson Goes,1 Dicken Ko,1 Waichi Wong,1 Robert B. Colvin,4 Megan Sykes,2 A. Benedict Cosimi.1 1Transplantation Unit, Massachusetts General Hospital, Boston, MA; 2Trasnplant Biology Research Center, MGH, Charlestown, MA; 3Bone Marrow Transplant Unit, MGH, Boston, MA; 4Pathology, MGH, Boston, MA; 5this Study was Performed in Partnership with Immune Tolerance Network. BACKGROUND: Induction of allograft tolerance has been the ultimate goal in organ transplantation. METHODS: Five subjects with ESRD received combined kidney and bone marrow transplantation (CKBMT) from HLA mismatched donors. The preparative regimen consisted of cyclophosphamide, thymic irradiation, anti-CD2 mAb and a 9-14 months course of a calcineurin inhibitor. Two subjects also received two doses of antiCD20 mAb prior to transplant. RESULTS: All recipients developed transient mixed chimerism without GVHD.Complete withdrawal of immunosuppressive medication was achieved in 4/5 recipients with current immunosuppression-free survival exceeding 3.2, 2.5, 1.1 and 0.2 years. A recipient rejected his kidney allograft on day 10. (1) The most recent creatinine/GFR of these 4 subjects were 1.2/61, 1.6/75, 1.6/96 (by renogram), 1.8/71 ml/min, respectively. (2)Although Subjects 2 and 5 initially developed anti-donor antibodies (ADA), both recipients lost ADA by 10th and 3rd month, respectively. Subject 4 developed post-transplant de novo anti-class II ADA one month after discontinuation of his immunosuppression, but without any functional derangements. (3) Subjects 1, 2 and 4 showed donor specifi c hyporesponsiveness in CML and MLR after nine months. (4) Allograft biopsies taken at 3 years from Subjects 1 and 2 revealed normal glomerulus, no tubulitis and no vasculopathy by light and electron microscopic examination with no C4d staining. Biopsy taken at 1.5 years from Subject 4 showed positive C4d deposition with no changes diagnostic of rejection. A most recent biopsy taken from Subject 5 on day 243 showed no rejection. (5) In Subject 1, extensive warts due to human polyoma virus existed before transplantation, but it disappeared after discontinuation of immunosuppression. All four patients show no hypertension, no DM, no abnormal lipid profi les. CONCLUSIONS: Renal allograft function of CKBMT recipients has been stable up to 3 years after complete withdrawal of their immunosuppression. Abstract# 3 A Novel Human Anti-CD40 Monoclonal Antibody, 4D11, for Kidney Transplantation in Cynomolgus Monkeys, Effect of Induction and Maintenance Therapy. Takeshi Aoyagi,1 Tomomi Suzuki,1 Atsushi Sugitani,2 Atsushi Imai,1 Motohiro Uno,1 Ryoichi Goto,1 Kenichiro Yamashita,1 Toru Miura,3 Nobuaki Takahashi,3 Tomoo Itoh,4 Hiroyuki Furukawa,1 Satoru Todo.1 11st Dept of Surgery, Hokkaido Univ., Sapporo, Hokkaido, Japan; 21st Dept of Surgery, Kyusyu Univ., Hakata, Fukuoka, Japan; 3Kirin Brewery Co., Ltd, Takasaki, Gunma, Japan; 4Dept of Surgical Pathology, Hokkaido Univ. Hospital, Sapporo, Hokkaido, Japan. Background/Aim: We have previously demonstrated that a novel human anti-CD40 monoclonal antibody (mAb), 4D11, markedly prolongs renal allograft survival in cynomolgus monkeys. In this study, we evaluated the effect of induction and maintenance therapy using 4D11. Methods: Twenty-four monkey renal transplants were divided into three groups: no treatment (Control), induction (Group A) and maintenance (Group B) treatments. Groups A and B were further subdivided according to the dose of 4D11 (n=3 each). The 4D11 was given intravenously on days 0, 4, 7, 11 and 14 for the induction therapy. In the group B, a half of the initial dose was given weekly thereafter until day 180. Graft survival, graft histology, anti-4D11 and anti-donor antibodies were examined. Results: Treatment Group 4D11 (mg/kg) Graft survival (days) Mean (days) Cause of death No treatment Control 5, 6, 7 6.0 AR, AR, AR Induction A-I 5 >96, 79, 75 83.3 Alive, AR, AR A-II 10 >200, 107, 92 133.0 Alive, CAN, AR A-III 20 169, 105, 90 121.3 AR, AR, AR Maintenance B-I 1 80, 79, 10 56.3 AR, AR, AR B-II 5 >202, 98, 44 114.3 Alive, AR, AR B-III 10 374, 253, 27 218.3 AR, AR, UTI B-IV 20 216, 169, 153 179.3 CAN, AR, CAN AR: Acute rejection, CAN: Chronic allograft nephropathy, UTI: Urinary tract infection No adverse events were observed. Graft survival was markedly prolonged by the induction treatment, which was further extended by the maintenance treatment. While graft biopsies revealed grade II or III rejection in the control, majority of those from groups A and B at 1 month post-transplantation showed only a borderline change. Anti 4D11-Ab was detected only in group A-III after drug cessation. In addition, animals receiving induction treatment and those given maintenance treatment with high doses of 4D11 did not develop anti-donor Ab unless the treatment was discontinued. Conclusion: 4D11 exhibited potent immunosuppressive effect on renal allograft survival both by induction and maintenance treatments. Besides, anti-drug and anti-donor antibody productions were effectively suppressed by 4D11. Abstract# 4 Pre-Transplant (Tx) Donor-Specifi c B Cells in Highly Sensitized Patients (HS), Detected by Intracellular Cytokine Flow Cytometry (CFC) Predict Antibody Mediated Rejection (AMR). Mieko Toyoda,1 Andy Pao,1 Ashley Vo,1 Marina Lukovsky,1 Raju Radha,1 Tetsu Sado,1 Lara Baden,1 Anna Petrosyan,1 Stanley C. Jordan.1 1Transplant Immunology Laboratory and Comprehensive Transplant Center, Cedars-Sinai Medical Center, Los Angeles, CA. Background: Intravenous immunoglobulin treatment (IVIG-Rx) reduces panel reactive antibodies and crossmatch (CMX) positivity, facilitating CMX(-) kidney Tx in HS. However, high rates of allograft rejec