Infusion of Lymphocytes Treated With 8-Methoxypsoralen and Ultraviolet A Light Induces CD19+IL-10+ Regulatory B Cells and Promotes Skin Allograft Survival.

Extracorporeal photopheresis (ECP) represents an alternative to immunosuppression as a means of reducing rejection after thoracic organ transplantation. The mechanism by which ECP exerts its protective effects, until now, has remained elusive. Infusion of ECP-treated splenic lymphocytes (PUVA-SP) can induce CD4+CD25highFoxp3+ regulatory T cells. However, the regulatory effect of PUVA-SP on B cells remains poorly understood. In the present study, we measured IL-10 secretion from CD19+ B cells of peripheral blood mononuclear cells. Our results demonstrate that infusion of PUVA-SP (PUVA-BSP from BALB/c or PUVA-CSP from C57BL/6 mice), in the absence of an immunosuppressant, significantly promotes skin allograft survival. This effect was associated with upregulation of circulating regulatory B cells exhibiting preferential IL-10 secretion and a shift of cytokine profile from helper T cell type 1 to helper T cell type 2. Our results suggest that effective treatments involving infusion of PUVA-SP is likely related not only to the modulation of T cell and regulatory T cell functions but also to the function of B cell and regulatory B cells.

A clinically feasible approach to induce delayed tolerance in recipients of prior kidney or vascularized composite allotransplants.

BACKGROUND: Mixed chimerism induces donor-specific tolerance to kidney and vascularized composite allotransplants (VCA). However, simultaneous kidney or VCA and bone marrow transplantation (BMT) is problematic because of the combined risk and time required for conditioning. Here, we developed a delayed tolerance induction strategy with mixed chimerism through BMT in prior kidney or VCA recipients. METHODS: Wistar Furth rats that received kidney transplantation (KTx) or VCA from allogeneic August-Copenhagen Irish donors were maintained on immunosuppression (IS) for 8 weeks. These recipients were then conditioned with anti-αß-T-cell receptor and anti-CD8 monoclonal antibodies, total body irradiation, cyclosporine A and mycophenolate mofetil (12 doses), and antilymphocyte serum (one dose); and transplanted with T-cell-depleted donor marrow. All IS was discontinued on day 11 after BMT. RESULTS: Cyclosporine A monotherapy prevented acute rejection of KTx or VCA. However, all allografts were rejected after IS withdrawal in KTx or VCA recipients who were conditioned but did not receive BMT. After delayed BMT, mixed chimerism was initially achieved in all KTx or VCA recipients with 200-, 300-, and 400-cGy total body irradiation. Long-term tolerance to KTx or VCA was achieved in most of these recipients with total IS withdrawal. The tolerance achieved with delayed BMT was donor specific as confirmed by acceptance of donor skin and rejection of third-party skin graft. CONCLUSIONS: IS-free donor-specific tolerance can be successfully induced with delayed BMT to previous recipients of kidney transplantation or VCA. These findings have significant clinical implications for transplant recipients who receive an organ from either a living donor or a deceased donor with frozen bone marrow cells available.

Myeloablative irradiation and granulocyte colony-stimulating factor prolong the survival of chimeric limb allografts in a rodent model.

BACKGROUND: Limb allografts consist of different types of tissues with varying antigenicities. One of the key components is the bone marrow. Removal and reconstitution of this tissue may allow better control of limb allograft rejection. The authors evaluated the efficacy of a new protocol for bone marrow removal that uses pretransplant graft irradiation, granulocyte colony-stimulating factor, and temporal FK506, with particular reference to the dose requirement and toxicity of irradiation. METHODS: In total, 57 whole-limb allotransplants from LacZ transgenic rats to Lewis rats were performed. Graft irradiation with various doses ranging from 250 to 1500 R was administered just before transplantation. Granulocyte colony-stimulating factor was given for 4 days, whereas FK506 was used for 28 days. The level of intra-bone marrow chimerism of the graft was evaluated by semiquantitative polymerase chain reaction. RESULTS: Rejection-free survival of grafts was increased significantly in groups that received more than 1000 R of pretransplant irradiation and granulocyte colony-stimulating factor, whereas grafts that received less than 500 R of irradiation showed no prolongation in survival. Bone marrow in the myeloablative irradiation groups was quickly reconstituted by recipient-derived cells. In the group treated with 1500 R of irradiation, the graft tissue was damaged and this led to infection of the recipient. In the 1000-R group, two of eight recipients showed rejection-free graft survival of more than 1 year without any immunosuppression. CONCLUSIONS: The authors study found that removal of allogeneic bone marrow in the limb graft and rapid reconstitution with recipient marrow cells reduced the recipient's immune response. Graft rejection was delayed significantly but not completely prevented.

Prolonged survival of experimental extremity allografts: a new protocol with total body irradiation, granulocyte-colony stimulation factor, and FK506.

The induction of a high-level of chimerism (macrochimerism) may be the most reliable strategy for achieving donor-specific tolerance. The purpose of this study was to evaluate the efficacy of a new protocol using total-body irradiation (TBI) and granulocyte-colony stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. In this study, we investigated whether the timing of TBI influenced the period of graft survival. In total, 50 whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. TBI was performed at days 0 and 14, and G-CSF was given for 4 days after TBI. FK506 was given for 28 days after transplant. Nontreated limb allografts were rejected after 4.2 days. The survival time was prolonged to 64 days in the FK506 monotherapy group. In the group receiving TBI at day 14, limb allograft survival was significantly prolonged to 81 days. In the group receiving TBI at day 0, 26% of recipients died but in the surviving recipients the grafts survived for longer than 1 year without lethal graft-versus-host disease (GVHD). Polymerase chain reaction (PCR) analysis revealed a high level of intrabone marrow chimerism in the recipient, thus demonstrating successful induction of macrochimerism. A new protocol of pretransplant TBI followed by treatment with G-CSF and FK506 was found to induce a high level of chimerism and to significantly prolong the survival of limb allografts in recipients without lethal GVHD.

Tolerance induction by third-party "off-the-shelf" CD4+CD25+ Treg cells.

OBJECTIVE: Recent reports have shown that donor or host CD4(+)CD25(+) Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. In the present study we investigated the potential of third-party Treg cells compared to donor-type cells to facilitate BM allografting. METHODS: Graft rejection is assessed in a mouse model of T cell-mediated BM allograft rejection. Lethally irradiated C3H mice are transplanted at day 2 after irradiation with T cell-depleted Balb/Nude BM. Graft rejection is induced by purified host-type T cells infused one day prior to BMT. Cells tested for their facilitating activity are added to the T cell-depleted BM allograft. RESULTS: Naïve or ex vivo-expanded third-party Treg cells can effectively enhance engraftment of T cell-depleted BM allografts, exhibiting reactivity in vitro and in vivo similar to that found for donor-type Treg cells. CONCLUSION: The use of third-party Treg cells in contrast to donor-type cells could allow advanced preparation of a large bank of Treg cells (off-the-shelf), with all the appropriate quality controls required for cell therapy.

Immune unresponsiveness by intraportal UV-B-irradiated donor antigen administration requires persistence of donor antigen in a nerve allograft model.

The purpose of this study was to characterize the mechanism of unresponsiveness produced by the intraportal administration of ultraviolet-B (UV-B)-irradiated donor antigen. Pretreated Buffalo rats accepted Lewis nerve allografts, had decreased in vitro T-cell reactivity, and demonstrated nerve regeneration and recovery of limb function, while rejecting third-party nerve allografts. Regenerated nerve grafts were then retransplanted into a second naïve recipient. Rejection of the retransplanted allograft by naïve donor-strain, but not recipient-strain, animals suggests that the allografts were completely replaced by host tissue. Pretreated Buffalo rats were also given a second Lewis allograft after the first had regenerated. The second allograft was rejected and in vitro immune reactivity was comparable to naïve animals. Because the unresponsiveness state was extinguished with loss of exposure to donor antigen, these findings suggest that the intraportal administration of UV-B-irradiated donor antigen works by anergic or suppressive regulatory, rather than deletional, mechanisms.

Psoralen and UVA light: an in vitro investigation of multiple immunological mechanisms underlying the immunosuppression induction in allograft rejection.

Photopheresis (ECP) is a novel immunomodulatory therapy effectively used to treat several T-cell-mediated diseases and to reverse allograft rejection after organ transplantation. It consists of infusion of UVA-irradiated autologous leukocytes collected by apheresis and extracorporeally incubated with 8-methoxypsoralen (8-MOP). In this study we explored the potential immunological events for therapeutic efficacy of photopheresis in preventing allograft rejection by evaluating in vitro the combined effects of 8-MOP and UVA (PUVA) on multiple immunological parameters, such as induction of apoptosis, production of soluble mediators, and expression of cell antigens. Peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects were treated with 8-MOP and UVA at the same doses as those clinically used in ECP. We demonstrate that PUVA treatment induced leukocyte hyporesponsiveness and a decrease in expression of co-stimulatory and adhesion molecules as well as of cytokine levels. Additionally, PUVA treatment induced apoptosis in both mononuclear cells (possibly through the Fas/FasL system and/or the CD38 pathway) and purified monocytes. In conclusion, our work focuses attention on the initial phase of immune response and identifies some new targets of therapy (e.g., costimulatory molecules) able to trigger final effects underlying therapeutic efficacy of photopheresis.

Portal venous ultraviolet B-irradiated donor alloantigen prevents rejection in circumferential rat tracheal allografts.

BACKGROUND: Before tracheal transplantation can be considered as a method of reconstruction in patients with extensive circumferential tracheal defects, we must achieve a state of nontoxic, donor-specific tolerance so that the risks of such a transplant do not outweigh the benefits. OBJECTIVE: Our objective was to determine whether a single intraportal injection of modified donor alloantigen achieves donor-specific immunosuppression for major histocompatibility complex-mismatched rat tracheal allografts. STUDY DESIGN: Buffalo (recipient) rats were pretreated with either a single portal-vein administration of ultraviolet B (UVB)-irradiated donor splenocytes (n = 4) or an intraportal inoculation of nonirradiated donor splenocytes (n = 4). Major histocompatibility complex-mismatched Lewis (donor) tracheal allograft segments were then grafted into treatment groups 7 days after donor-cell pretreatment. Tracheal rejection was assessed by histologic analysis, mucosal cilia motility, and in vitro immunologic assessment. RESULTS: The UVB-treated group demonstrated no acute or chronic rejection as well as complete functional recovery. In vitro immunologic assessment demonstrated a donor-specific hyporesponsiveness and donor allospecificity. Untreated animals and those receiving nonirradiated donor splenocytes showed acute rejection of their tracheal allografts. CONCLUSION: Recipient pretreatment with intraportally administered UVB-irradiated donor splenocytes prevents rejection of circumferential rat tracheal allograft segments by inducing a donor-specific immune hyporesponsiveness.

Pretreatment with portal venous ultraviolet B-irradiated donor alloantigen promotes donor-specific tolerance to rat nerve allografts.

OBJECTIVE: To determine if a single intraportal inoculation of ultraviolet B-irradiated (UVB) donor splenocytes can prevent nerve allograft rejection and confer donor-specific immunotolerance to rat nerve allograft segments. METHODS: Age-matched, class I and class II major histocompatibility complex (MHC) mismatched Buffalo (RT1b) rats were transplanted with a syngeneic nerve isograft, a Lewis (RT1l) nerve allograft, or a Brown-Norway (RT1n) rat nerve allograft segment. Control Buffalo rats in group I received a 3.0-cm Lewis (RT11) sciatic-posterior tibial interposition nerve allograft without pretreatment; group II Buffalo rats received a syngeneic Buffalo nerve isograft without pretreatment. Group III Buffalo recipients were inoculated with 2.5 x 107 UVB-irradiated Lewis donor splenocyte cells by portal venous administration 7 days before transplantation with a 3.0-cm sciatic-posterior tibial nerve allograft from a Lewis (RT11) or a third party Brown-Norway rat (RT1n) donor (group IV). Nerve graft regeneration was assessed with walking track analysis, nerve conduction studies, retrograde neural tracing, nerve graft histology, and morphometry. Recipient immune tolerance was assessed through in vitro immunological assessment. RESULTS: Pretreatment with UVB-irradiated donor splenocytes 7 days before transplantation prevented nerve allograft rejection. Pretreated animals receiving a nerve allograft recovered limb function, and demonstrated morphological, histological, and electrophysiologic parameters of nerve regeneration similar to that measured in rats receiving a nerve isograft. In vitro immunological assessment by mixed lymphocyte culture (MLC), cytotoxic T lymphocyte (CTL) assay, limiting dilution analysis (LDA) of helper (pTH) and cytotoxic (pCTL) precursor frequencies, and IL-2 production demonstrated a marked donor-specific suppression in allografted animals pretreated with intraportal UVB-irradiated donor splenocytes. These assessments correlated with indefinite acceptance of donor nerve allografts. CONCLUSIONS: A single pretreatment with a single intraportal dose of UVB-modified donor antigen specifically induces tolerance to peripheral nerve allografts in rats.

CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway.

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.