A clinically relevant CTLA4-Ig-based regimen induces chimerism and tolerance to heart grafts

A Clinically Relevant CTLA4-Ig-Based Regimen Induces Chimerism and Tolerance to Heart Grafts Sen Li, MD, Mohan Thanikachalam, MD, Manhui Pang, MD, Nobuyoshi Kawaharada, MD, Abdelouahab Aitouche, PhD, and Si M. Pham, MD Division of Cardiothoracic Surgery, University of Miami School of Medicine, Miami, Florida, and Division of Cardiothoracic Surgery, Sapporo Medical University, Sapporo, Japan

Background. We determined whether a nontoxic CTLA4-Ig-based conditioning regimen effected mixed chimerism and donor-specific tolerance when heart and bone marrow were transplanted simultaneously. Methods. Fully mismatched rat strain combinations were used. Recipients received total-body irradiation (300 centigrays), bone marrow (108 cells), and cardiac transplants from the donor on day 0. Subsequently, recipient animals received CTLA4-Ig (2 mg/kg, every other day, ⴛ 5 doses), tacrolimus (1 mg/kg/day; days 0 to 9), and one dose (10 mg) of antilymphocyte serum on day 10. Results. All bone marrow recipients (n ⴝ 7) developed mixed chimerism (mean ⴝ 25% ⴞ 9% at 1 year) and

accepted cardiac allografts permanently (> 375 ⴞ 32 days). Recipients that received conditioning regimen but no bone marrow (n ⴝ 5) rejected donor hearts within 51 ⴞ 13 days (p < 0.01). Recipients that accepted heart grafts also permanently accepted (> 180 days) donorspecific skin grafts, but rapidly rejected (< 10 days) third-party skin grafts. Conclusions. A nontoxic CTLA4-Ig-based conditioning regimen effects mixed chimerism and donor-specific tolerance when heart and bone marrow are transplanted simultaneously. This regimen may have clinical application. (Ann Thorac Surg 2001;72:1306 –10) © 2001 by The Society of Thoracic Surgeons

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Material and Methods

rgan transplantation between genetically disparate individuals currently requires the use of immunosuppressive agents to suppress rejection. The deleterious side effects of these agents and their inability to prevent rejection completely have led to a continuing search for methods to induce donor-specific transplantation tolerance. Donor-specific transplantation tolerance is a state in which the host permanently accepts the transplanted organ without antirejection drugs, yet is able to retain immunocompetence so that infection, malignancy, and end-organ toxicities related to nonspecific immunosuppression can be avoided. It has been demonstrated that mixed hematopoietic chimerism (MC), a state in which bone marrow stem cells of different genetic background coexist, is associated with donor-specific transplantation tolerance [1]. However, the toxicity associated with conditioning regimens required to achieve bone marrow engraftment has limited the clinical application of MC. In this study, we demonstrate a clinically relevant CTLA4Ig-based nontoxic conditioning regimen that effects mixed chimerism and donor-specific tolerance when heart and bone marrow are transplanted simultaneously.

Presented at the Thirty-seventh Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 29 –31, 2001. Address reprint requests to Dr Pham, Division of Cardiothoracic Surgery, University of Miami School of Medicine, Highland Professional Building, 1801 N W 9th Ave, 5th Floor, Miami, FL 33136; e-mail: spham@ med.miami.edu.

© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

Animals Six- to 8-week-old major histocompatibility complex (MHC) and minor antigen-mismatched Wistar Furth (WF; RT1.Au), August Copenhagen Irish (ACI; RT1.Aa), and Lewis (RT1.A1) rats were purchased from Harlan Sprague Dawley (Indianapolis, IN) and housed in a pathogen-free facility. All animals were treated in compliance with the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences, and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).

Experimental Design Fully mismatched 4 to 6-week-old ACI and Wistar Furth (WF) rats were used as donors and recipients, respectively. On the day of transplantation (day 0), T-celldepleted bone marrow from ACI donors were prepared. WF recipients were subjected to total-body irradiation (TBI; 300 centigrays [cGy]) and bone marrow infusion (108 bone marrow cells per animal) via penile veins. Unfractionated TBI was performed with a cesium-137 source (Gamma-cell; Nordion, Ontario, Canada). Subsequently, the recipient animals underwent heterotopic heart transplantation. After transplantation, recipients received tacrolimus (1 mg/kg/day, intramuscularly; Fujisawa Pharmaceutical Co, Japan) from day 0 to day 9, human CTLA4-Ig (2 mg/kg, intraperitoneally; a gift from 0003-4975/01/$20.00 PII S0003-4975(01)03066-1

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Robert J. Peach, MD; Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ) on days 0, 2, 4, 6, and 8, and one dose (10 mg/rat, intraperitoneally) of antilymphocyte serum (ALS; Accurate Chemical Co, Westbury, NY) on day 10. In the treatment control group, all recipient animals received the same conditioning regimen except donor bone marrow. In the naı¨ve control group, naı¨ve WF recipients received ACI heart, and no other treatment. Bone marrow recipients with longsurviving (⬎ 120 days) cardiac grafts received challenging donor-specific (ACI), third-party (Lewis), and syngeneic (WF) skin grafts, 120 to 150 days after heart transplantation without additional immunosuppression.

Preparation of T-Cell-Depleted Bone Marrow Cells Marrow donors were euthanized, and marrow was flushed from femurs and tibias and resuspended in medium 199 (Gibco, Grand Island, NY) containing 2 ␮g/mL gentamicin. Marrow was filtered through a sterile nylon mesh, washed at 1,200 rpm for 5 minutes, resuspended, and counted. T-cell depletion was accomplished by incubating untreated marrow with monoclonal antibody (mAbs) against ␣␤-T-cell receptor (␣␤-TCR) (R73; mouse IgG1; Serotec, Washington, DC) at 4°C for 30 minutes. Residual antibody was removed by washing twice in RPMI medium containing 2% fetal calf serum (Gibco). Depletion was accomplished by incubating marrow cells with immunomagnetic beads coated with sheep anti–mouse IgG (M-450; Dynabeads, Lake Success, NY) at a bead to cell ratio of 1:4 for 60 minutes at 4°C. Separation was achieved by applying a hand-held magnet to the test tube for 3 minutes followed by careful elution of the nonadherent cell solution. Cells were then resuspended in medium 199 and standardized to a concentration of 108 cells/mL and injected into the recipients via penile veins. Efficiency of T-cell depletion was confirmed with flow cytometric analysis using fluorescein isothiocyanate (FITC)-labeled goat anti–mouse IgG1 (Southern Biotechnology Associates, Inc, Birmingham, AL), a secondary antibody to mouse anti-␣␤-TCR mAbs.

Determination of Bone Marrow Engraftment by Flow Cytometry All bone marrow recipients were assessed 30 days after reconstitution for the presence of donor engraftment. Whole blood was collected in heparinized tubes and directly labeled with anti-WF (recipient) or anti-ACI (donor) class I biotinylated mAbs (a gift from Drs Heinz W. Kunz and T. J. Gill III; University of Pittsburgh School of Medicine, Pittsburgh, PA) for 30 minutes at 4°C. Cells were then washed with fluorescence-activated cell sorter (FACS) solution and counterstained with streptavidinFITC (Pharmingen, San Diego, CA) for 15 minutes. Red cells were lysed by the addition of lysing buffer (FACS lysing solution; Becton Dickinson, San Jose, CA) for 10 minutes. Cells were resuspended in 2% paraformaldehyde. Flow cytometry was done on a FACSort (Becton Dickinson, San Jose, CA). The level of donor chimerism was calculated based on positive staining above an inflection point chosen to maximize staining of positive cell

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populations while minimizing staining of a concurrent control cell population.

Assessment of Graft-Versus-Host Disease (GVHD) Animals were weighed at the time of transplant and at varying intervals thereafter. Clinical assessment of GVHD was based on the characteristic appearance of GVHD in rats, including unkempt appearance, hair loss, diarrhea, rash on paws, snout, and skin, and failure to thrive [2]. Clinical GVHD was graded as absent, mild, and severe. At the time of sacrifice, sections of skin and tongue were stained with hematoxylin and eosin (H & E) and examined for the presence of dermal lymphoid infiltration, subepidermal cleft formation, or loss of epidermis, indicative of GVHD [2].

Heterotopic Cardiac Transplantation Heterotopic cardiac transplantation was performed within 1 to 4 hours after bone marrow transplantation, as previously described [3]. Briefly, ACI hearts were procured and stored in a cold saline bath. Recipients were anesthetized with methoxyflurane, and the aorta and inferior vena cava exposed through a midline laparotomy. Arterial anastomosis between donor aorta and recipient aorta and venous anastomosis between donor pulmonary artery and recipient inferior vena cava were constructed with 8-0 monofilament sutures. Allograft survival was evaluated by daily palpation and graded on a scale of ⫹4 (strong pulse) to 0 (no pulsation).

Skin Grafting Skin grafting was performed as previously described [4]. Full-thickness tail skin grafts were harvested from syngeneic (WF), third-party (Lewis), and donor-specific (ACI) rats and grafted to the dorsal thoracic wall of chimeric recipients that had accepted cardiac grafts for more than 120 days. Grafts were visually assessed for rejection, which was considered complete when no viable skin was present.

Statistical Analysis Continuous variables are expressed as mean ⫾ standard error of the mean (SEM). Graft survivals between groups were compared using the Mann-Whitney U test. Differences were considered significant when p was less than 0.05. All statistical analyses were performed using Statistica software package (1998 edition; Statsoft, Inc, Tulsa, OK).

Results Stable Mixed Hematopoietic Chimerism and Robust Donor-Specific Tolerance to Heart Allografts One hundred percent (7 of 7) of animals conditioned with the CTLA4-Ig-based regimen developed MC. The MC was stable with a mean donor chimerism of 25% ⫾ 9% at 1 year (Table 1). There was no conditioning-related morbidity or mortality. None of the animals showed clinical or histological evidence for acute or chronic

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Table 1. Mixed Chimerism and Donor-Specific Tolerance to Heart Allografts Group

n

Recipient (WF) Treatment

1 2 3

5 5 6

None CTLA4-Ig, FK, ALG, TBI CTLA4-Ig, FK, ALG, TBI ⫹ BMb

Heart Donorsa

Donor Chimerism at 30 Days

ACI ACI ACI

0% 0% 18% ⫾ 6% 25% ⫾ 9%c

Survival (Days) 7, 7, 8, 8, 8 75, 43, 45, 51, 45 ⬎328, ⬎328, ⬎399, ⬎399, d ⬎390, ⬎390, ⬎390

CTLA4-Ig ⫽ 2 mg/kg/day, every other day (days 0 to 8); FK ⫽ Tacrolimus, 1 mg/kg/day, every day (days 0 to 9); ALG ⫽ anti-lymphocyte globulin, 10 mg, single dose (day 10); TBI ⫽ total-body radiation, 300 cGy (day 0). b Heterotopic heart transplants on day 0. BM ⫽ Bone marrow cells. 100 million T-cell-depleted ACI bone marrow cells (day 0). d at 1 year. p ⬍ 0.01 vs groups 1 and 2 (Mann-Whitney U test).

a

c

Chimerism

GVHD. All bone marrow recipients (n ⫽ 7) exhibited donor-specific tolerance to cardiac allografts (mean graft survival time: ⬎ 375.0 ⫾ 32.0 days; Table 1, group 3). In contrast, all naı¨ve (group 1, n ⫽ 5) and treatment controls (group 2, n ⫽ 5) rejected ACI hearts within 7.6 ⫾ 0.2 and 51.0 ⫾ 13.0 days, respectively (p ⬍ 0.01; group 3 vs groups 1 and 2; Mann-Whitney U test).

Tolerance of Second-Set Donor-Specific Skin Grafts Recipients with long-surviving (⬎ 120 days) cardiac grafts received challenging donor-specific (ACI), thirdparty (Lewis), and syngeneic (WF) skin grafts, 120 to 150 days after heart transplantation without additional immunosuppression. Mean survival time for third-party challenging skin grafts (n ⫽ 4) was 12 days. In striking contrast, all syngeneic (n ⫽ 4) and donor-specific (n ⫽ 4) grafts were accepted for more than 180 days (n ⫽ 4) (p ⬍ 0.01 vs third-party grafts; Fig 1). Long-term accepted skin grafts demonstrated normal texture and hair growth (Fig 2).

Fig 1. Survival of challenging skin grafts placed in chimeras tolerant more than 120 days to donor-specific hearts. Full-thickness tail skin grafts were harvested from third-party (Lewis) and donor-specific (ACI) rats and grafted to the dorsal thoracic wall of chimeric recipients. Mean survival time for third-party skin grafts (n ⫽ 4) was 12 days, while all donor-specific (n ⫽ 4) grafts exhibited complete acceptance for more than 180 days (n ⫽ 4) (p ⬍ 0.01 vs thirdparty grafts).

Fig 2. (A) A challenging donor-specific skin graft 180 days after transplantation into a mixed chimera that had accepted a donorspecific heart allograft for 120 days. Skin graft shows normal texture and hair growth. (B) Challenging third-party skin graft 8 days after transplantation into a mixed chimera that had accepted a donorspecific heart allograft for 120 days. There is no viable skin graft due to acute rejection.

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Comment In this study, we have demonstrated that a nontoxic CTLA4-Ig-based conditioning regimen effects mixed chimerism and donor-specific tolerance when heart and bone marrow are transplanted simultaneously. The conditioning regimen consists of CTLA4-Ig (which blocks the CD28:B7 costimulatory pathway), tacrolimus, antithymocyte globulin, and low-dose (300 cGy) total-body irradiation. This conditioning regimen was well tolerated by the animal. No animal developed acute or chronic GVHD. All animals that received donor bone marrows developed robust donor-specific transplantation tolerance; recipients with long-term surviving cardiac grafts accepted a challenging donor-specific skin graft while promptly rejected a third-party skin graft. One unique feature of this conditioning regimen is that the donor bone marrow was transplanted at the time of cardiac transplantation as opposed to weeks or months before, as reported in previous studies [5, 6]. This regimen may be relevant in clinical transplantation, in which the majority of organs are from cadaveric donors. Mixed chimerism was first achieved in adult mice by Ildstad and Sachs [4]. In this model, a mixture of T-celldepleted (TCD) syngeneic plus allogeneic bone marrow was used to reconstitute lethally irradiated (950 cGy) mice. Over the past decade, MC has been achieved in various animal models and has consistently led to tolerance to different organs [7]. However, the toxicity from the conditioning regimen, and GVHDs associated with allogeneic bone marrow transplantation, has limited the application of MC in clinical transplantation. In bone marrow transplantation, irradiation functions to eliminate donor-reactive T-cells in the recipient (thus prevents rejection), and to make “space” for the transplanted stem cells to engraft [1]. We have previously demonstrated that adjuvant treatment of the recipient with ALS and tacrolimus reduces the dose of TBI (from 1,000 to 500 cGy) required to achieve mixed chimerism and tolerance to cardiac allografts in fully mismatched rats [3]. Tacrolimus blocks the T-cell receptor (TCR)-mediated pathway of T-cell activation at the level of calcineurin phosphatase [8]. Cyclosporine A (CysA), which has a similar mechanism of action as tacrolimus, also enhances allogeneic bone marrow engraftment [9, 10]. In a recent study, to further inhibit recipient’s T-cell activation, we added costimulatory blockade with CTLA4-Ig to our previous regimen of tacrolimus and ALS. This combination further reduced the dose of radiation (to 300 cGy) required for bone marrow engraftments in fully disparate rats [11]. CTLA4-Ig is a fusion protein that blocks the CD28:B7 costimulatory pathway, which is essential for full T-cell activation. The activation of naı¨ve T-cells requires two signals: a primary signal, which is initiated by the binding of T-cell receptors (TCR) with major histocompatibility antigen complex presented on the antigen-presenting cells (APCs), and a secondary costimulatory signal [12]. Among costimulatory signals that have been described, the CD28:B7 is an important one. The binding of CD28 molecules, which are ex-

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pressed on T-cells, to their ligands (B7-1 and B7-2 molecules) expressed primarily on APCs, delivers a powerful costimulatory signal for T-cell activation. CTLA4-Ig binds B7 molecules with higher affinity than CD28 and, therefore, acts as a competitive inhibitor of CD28/B7-mediated T-cell costimulation [13]. Initial studies using costimulatory blockage alone (without donor bone marrow) to prolong the survival of organ allografts had shown great promise. Larsen and associates reported long-term acceptance of heart and skin allografts in mice by blocking both CD28 (with CTLA4-Ig) and CD40 (with MR1 antibody) costimulatory pathways [14]. These data were duplicated in rhesus monkeys by Kirk and associates, who demonstrated that a combination of CTLA4-Ig and CD40L-specific mAbs (5C8) prolonged the survival renal allografts to more than 150 days in 2 of 4 treated animals [15]. However, subsequent studies have indicated that transplantation of allogeneic tissues under the cover of anti-CD40L mAbs has resulted in prolonged graft survival but not tolerance [16]. Honey and associates have demonstrated that failure to induce tolerance by costimulatory blockage probably results from the inability of anti-CD40L Abs to prevent graft rejection elicited by the cytotoxic (CD8⫹) T-cells [16]. Although not proven in this study, available data [6, 11] suggest that the most likely mechanism by which the CTLA4-Ig-based condition regimen leads to donor-specific tolerance of cardiac allografts in the current study is by allowing donor bone marrow cells to engraft as mixed hematopoietic chimerism. While toxicity associated with the conditioning regimens has been one of the major limitations to the application of mixed hematopoietic chimerism to induce tolerance in clinical transplantation, another major limitation is the GVHD associated with allogeneic bone marrow transplantation. Unlike humans and rats, mice do not develop GVHD even when unmodified allogeneic bone marrow is transplanted [6]. In rats, transplantation of unmodified bone marrow is associated with severe GVHD in up to 100% of the animal [17]. The T-cell compartment of the bone marrow contains a subset of T-cells with ␣␤-T-cell receptors (␣␤-TCR⫹) that is responsible for GVHD, as well as a separate subset of cells that have no ␣␤-T-cell receptors (␣␤-TCR⫺) known as facilitating cells [18, 19]. It has been demonstrated that facilitating cells markedly enhance bone marrow stem cell engraftment without causing GVHD [18]. Based on these data, we have elected to use anti-␣␤ TCR mAb for T-cell depletion in the current study to eliminate alloreactive donor T-cell clones responsible for GVHD, while preserving the facilitating cell population in the bone marrow graft. We have demonstrated that this T-cell depletion technique did not reduce the rate of bone marrow engraftment [20]. Because rats are more similar to humans in their propensity to develop GVHD, the use of T-cell depleted bone marrow, and the CTLA4-Ig-based conditioning regimen described in this study, may be more clinically relevant than those described in mice [5, 6]. In summary, we have demonstrated that a nontoxic CTLA4-Ig-based conditioning regimen that includes an-

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tithymocyte globulin, CTLA4-Ig, short course of tacrolimus, and low-dose (300 cGy) total-body irradiation effects mixed chimerism and donor-specific tolerance when heart and bone marrow are transplanted simultaneously. This clinically relevant conditioning regimen is well tolerated by the animal. This work was supported in part by grants from the American Heart Association (National Center; 960144590) and the Thoracic Surgery Research Foundation Fellowship Award to Mohan Thanikachalam.

References 1. Sykes M, Sachs DH. Mixed allogeneic chimerism as an approach to transplantation tolerance. Immunol Today 1988; 9:23–7. 2. Vallera DA, Blazar BR. T cell depletion for graft-versus-host disease prophylaxis. A perspective on engraftment in mice and humans. Transplantation 1989;47:751– 60. 3. Gammie JS, Li S, Zeevi A, Demetris AJ, Ildstad ST, Pham SM. Tacrolimus-based partial conditioning produces stable mixed lymphohematopoietic chimerism and tolerance for cardiac allografts. Circulation 1998;98:II163–9. 4. Ildstad ST, Sachs DH. Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts. Nature 1984;307: 168–70. 5. Hale DA, Gottschalk R, Umemura A, Maki T, Monaco AP. Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation. Transplantation 2000;69:1242–51. 6. Wekerle T, Sayegh MH, Ito H, et al. Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a nonmyeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance. Transplantation 1999;68:1348–55. 7. Gammie JS, Pham SM. Simultaneous donor bone marrow and cardiac transplantation: can tolerance be induced with the development of chimerism? Curr Opin Cardiol 1999;14: 126–32. 8. Flanagan WM, Corthesy B, Bram RJ, Crabtree GR. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 1991;352:803–7.

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9. Storb R, Yu C, Wagner JL, et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 1997;89: 3048–54. 10. Wagner JE. Prevention of graft failure by cyclosporine in rats receiving lymphocyte-depleted MHC-mismatched bone marrow. Transplantation 1992;53:624– 8. 11. Li S, Thanikachalam M, Pang M, Carreno M, Aitouche A, Pham SM. Combined host-conditioning with CTLA4-Ig, tacrolimus, anti-lymphocyte serum and low dose radiation leads to stable mixed hematopoietic chimerism in rats. Exp Hematol 2001;29:534– 41. 12. Schwartz RH. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 1992;71:1065– 8. 13. Linsley PS, Greene JL, Tan P, et al. Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. J Exp Med 1992;176:1595– 604. 14. Larsen CP, Elwood ET, Alexander DZ, et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 1996;381:434– 8. 15. Kirk AD, Harlan DM, Armstrong NN, et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA 1997;94:8789–94. 16. Honey K, Cobbold SP, Waldmann H. CD40 ligand blockade induces CD4⫹ T cell tolerance and linked suppression. J Immunol 1999;163:4805–10. 17. Colson Y, Zadach K, Nalesnik M, Ildstad S. Mixed allogeneic chimerism in the rat: donor-specific transplantation tolerance and prevention of chronic rejection for primarily vascularized cardiac allografts. Transplantation 1995;60:971– 80. 18. Kaufman C, Colson Y, Wren S, Watkins S, Simmons R, Ildstad S. Phenotypic characterization of a novel bonemarrow derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 1994;84:2436– 66. 19. Schuchert MJ, Wright RD, Colson YL. Characterization of a newly discovered T-cell receptor beta-chain heterodimer expressed on a CD8⫹ bone marrow subpopulation that promotes allogeneic stem cell engraftment. Nature Med 2000;6:904–9. 20. Neipp M, Exner BG, Maru D, et al. T-cell depletion of allogeneic bone marrow using anti-alphabetaTCR monoclonal antibody: prevention of graft-versus-host disease without affecting engraftment potential in rats. Exp Hematol 1999;27:860–7.

DISCUSSION DR RALPH DAMIANO (St. Louis, MO): That was a beautiful presentation. In your abstract, you described this as a “nonlethal” conditioning regimen. What could you tell us about some of the side effects of CTLA4-Ig, and what may be some of the roadblocks to using this in a clinical setting? DR THANIKACHALAM: One of the major complications of costimulatory blockade is thromboembolic problem. This complication has been reported in patients treated with the humanized anti-CD154 monoclonal antibody (Antova, hu5c8; Biogen, Inc.), resulting in the cessation of clinical trials in the areas of autoimmunity and transplantation. The main reason is that the

endothelial cells express the same ligands that the costimulatory blocker binds. This complication has not been reported with the use of CTLA4-Ig in clinical trial. In a recent trial in which CTLA4-Ig was used for the treatment of psoriasis (Abrams JR, Lebwohl MG, Guzzo CA, et al. CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris. J Clin Invest 1999;103:1243–52), the most common adverse events reported were uncomplicated upper respiratory tract infection and transient headache. There was no thromboembolic complication. Overall, the side effect of CTLA4-Ig in clinical transplantation remains unknown.