IL-5 and eosinophils mediate the rejection of fully histoincompatible vascularized cardiac allografts: regulatory role of alloreactive CD8(+) T lymphocytes and IFN-gamma

CD8(+) T lymphocytes are known to inhibit the development of eosinophilia and IL-5 synthesis in models of experimental lung disease. In transplantation, the rejection of fully mismatched cardiac allografts by recipients depleted of CD8(+) T cells is characterized by the recruitment of eosinophils in the rejected organs. We show here that this intragraft eosinophilia is dependent on the production of IL-5 since hearts transplanted into IL-5-deficient recipients depleted of CD8(+) cells did not contain eosinophils. More importantly, allograft survival was significantly extended in these animals. In mixed lymphocyte cultures (MLC), the presence of CD8(+) T cells in the responding cell population inhibited the secretion of IL-5. This inhibition was IFN-gamma dependent since adding neutralizing anti-IFN-gamma antibodies induced the production of IL-5. Furthermore, spleen cells isolated from IFN-gamma receptor (IFN-gammaR)-deficient mice secreted IL-5 upon allogeneic stimulation in primary MLC. In vivo, eosinophilia was observed in allografts rejected by IFN-gammaR-deficient recipients. On the contrary, grafts rejected by IFN-gammaR-deficient mice treated with neutralizing anti-IL-5 antibodies did not exhibit eosinophilic infiltration. Our study reveals the capacity of IL-5-secreting CD4(+) T cells and eosinophils to promote the rejection of heart allograft and demonstrates the importance of CD8(+) T cells and IFN-gamma in regulating this pathway of rejection.     

Tolerance induction towards cardiac allografts under costimulation blockade is impaired in CCR7-deficient animals but can be restored by adoptive transfer of syngeneic plasmacytoid dendritic cells

Deficiency of transplant recipients for the chemokine receptor CCR7 was originally described to slightly increase the survival time of vascularized solid organ grafts, probably due to a reduced priming of alloreactive T cells. Using a model of allotolerance induction by donor-specific splenocyte transfusion (DST) in combination with anti-CD40L mAb-mediated costimulation blockade (CSB), we show here a striking failure of CCR7-deficient (CCR7(-/-) ) recipients to tolerate cardiac allografts. Furthermore, in addition to the recently described lack of Treg, CCR7(-/-) mice were found to harbor significantly reduced numbers of plasmacytoid dendritic cells (pDCs) within peripheral as well as mesenteric lymph nodes (LNs), but not the bone marrow or spleen. pDCs had previously been suggested to function as tolerogenic APC during allograft transplantation, and a single transfer of syngeneic WT pDCs, but not conventional DCs, was indeed sufficient to rescue graft survival in DST+CSB-treated CCR7(-/-) recipients in a dose-dependent manner. We therefore conclude that the nearly complete absence of pDCs within LNs of CCR7(-/-) mice prevents the successful induction of DST+CSB-mediated allotolerance, leading to the observed acute rejection of cardiac allografts under tolerizing conditions.     

Anti-CCL25 Antibody Prolongs Skin Allograft Survival by Blocking CCR9 Expression and Impairing Splenic T-Cell Function

Chemokines, by virtue of their ability to recruit immune cells into allografts, play critical roles in acute transplantation rejection. CCR9 and its ligand, CCL25, is one of the key regulators of thymocyte migration and maturation in normal and inflammatory conditions. Moreover, several studies have revealed that high expression of CCR9 and CCL25 participated in many kinds of diseases. However, the role of CCR9 in allograft rejection is still unclear. In this study, we established a murine skin transplantation model of acute rejection. Our findings showed that the proportion of CCR9-expressing T cells was significantly increased in the spleen of allotransplanted mice compared with syngeneic transplantation. Furthermore, expression of CCL25 in allograft was similarly increased. Neutralization of CCL25 by intravenous injection of anti-CCL25 monoclonal antibody significantly prolonged skin allograft survival, decreased the number of infiltrating cells, and simultaneously suppressed the chemotactic ability and the proliferation of the splenic T cells in response to allogeneic antigens. Finally, blockade of CCL25 also diminished the secretion of IFN-γ by splenic T cells. These studies indicated that CCR9/CCL25 was involved in acute transplantation rejection and anti-CCL25 strategies might be useful in preventing acute rejection.     

Graft rejection mediated by CD4+ T cells via indirect recognition of alloantigen is associated with a dominant Th2 response

CD4(+) T cells that respond to indirectly presented alloantigen have been shown to mediate chronic rejection, however, the role of the indirect pathway in acute rejection has yet to be completely elucidated. To this end, BALB/c or C57BL/6 mice were depleted of CD8(+) T cells and transplanted with class II transactivator (CIITA)-deficient cardiac allografts, which cannot directly present class II alloantigens to CD4(+) T cells. In this manner, the rejection response by CD4(+) cells was forced to rely upon the indirect recognition pathway. When not depleted of CD8(+) cells, both BALB/c and C57BL/6 mice rejected CIITA-/- allografts and a polarized Th1 response was observed. In contrast, when BALB/c recipients of CIITA-/- allografts were depleted of CD8(+) T cells, the grafts were acutely rejected and a strong Th2 response characterized by eosinophil influx into the graft was observed. Interestingly, CD8-depleted C57BL/6 recipients of CIITA-/- allografts did not acutely reject their transplants and a Th2 response was not mounted. These findings indicate that CD4(+) T cells responding to indirectly presented alloantigens mediate graft rejection in a Th2-dominant manner, and provide further evidence for the role of Th2 responses in acute graft rejection.     

Prolonged skin allograft survival by IL-10 gene-introduced CD4 T cell administration

Both CD4 and CD8 T cells play crucial roles in immune responses in transplantation. Immunosuppressive drugs, such as FK506 and cyclosporin A, block the priming of alloreactive CD4 T(h) cells and the subsequent induction of allospecific CD8 cytotoxic effector T cells and inhibit allograft rejection. However, the desire to minimize chronic complications that may arise from the use of immunosuppressive agents drives the search for additional strategies for immunosuppression of allograft rejection. In this study, CD4 or CD8 T cells into which the IL-10 gene is introduced using an adenovirus vector containing human IL-10 (hIL-10) cDNA (Ad-hIL-10) and into mouse T cells transgenic for the Coxsackie virus and adenovirus receptor form a model system to study the effect of administration of IL-10-secreting T cells on the survival of the allogenic skin grafts. Ad-hIL-10-infected CD4 and CD8 T cells secreted a large amount of hIL-10 for 3-4 days in culture in vitro. Ad-hIL-10-infected CD4 T cells administered in vivo could be detected in the spleen for 7 days post-transfer. Significantly prolonged survival of grafts was observed in animals that received either Ad-hIL-10-infected activated CD4 T cells or T(h)2-skewed CD4 T cells as compared with controls. Furthermore, substantial enhancement of the effect was observed in B6.C-H2(bm1)/ByJ transplants. Thus, a direct manipulation of T cells through the introduction of the immunosuppressive cytokine gene IL-10 may be a novel strategy for the control of allograft rejection.     

Islet Transplantation in Mice Differing in the I and S Subregions of the H-2 Complex

Pancreatic islets from A.TH mice were transplanted into the spleen or streptozotocin (SZ)-diabetic A.TL mice. The two strains of mice are congenic inbred strains, differing only in the I and S subregions of the H-2 complex. The allogeneic islet grafts decreased blood glucose temporarily, but the islets were rejected after 21 ± 7 days (mean ± SD). The effect of skin presensitization was tested by giving both allogeneic and syngeneic skin grafts to each of a second set of A.TL mice before streptozotocin treatment and islet transplantation. The time course of rejection of the allogeneic islets in animals that received initial skin grafts was decreased to 8 ± 3 days. In both skin-presensitized and non-presensitized mice syngeneic islet grafts were able to restore normoglycaemia, even in animals that had previously rejected an islet allograft. These observations demonstrate that transplantation of pancreatic islet allografts across the I and S subregions of the H-2 complex is sufficient to induce rejection of the islets. The islet rejection was markedly accelerated by prior sensitization with allogeneic skin grafting. It is suggested that elements in allogeneic skin grafts serve as inducers of cytotoxic T-cell responses directed against gene products of the I and/or S subregions present on cells in the allogeneic islets.     

Induction of transplantation tolerance converts potential effector T cells into graft-protective regulatory T cells

Naturally occurring FOXP3(+) CD4(+) Treg have a crucial role in self-tolerance. The ability to generate similar populations against alloantigens offers the possibility of preventing transplant rejection without indefinite global immunosuppression. Exposure of mice to donor alloantigens combined with anti-CD4 antibody induces operational tolerance to cardiac allografts, and generates Treg that prevent skin and islet allograft rejection in adoptive transfer models. If protocols that generate Treg in vivo are to be developed in the clinical setting it will be important to know the origin of the Treg population and the mechanisms responsible for their generation. In this study, we demonstrate that graft-protective Treg arise in vivo both from naturally occurring FOXP3(+) CD4(+) Treg and from non-regulatory FOXP3(-) CD4(+) cells. Importantly, tolerance induction also inhibits CD4(+) effector cell priming and T cells from tolerant mice have impaired effector function in vitro. Thus, adaptive tolerance induction shapes the immune response to alloantigen by converting potential effector cells into graft-protective Treg and by expanding alloreactive naturally occurring Treg. In relation to clinical tolerance induction, the data indicate that while the generation of alloreactive Treg may be critical for long-term allograft survival without chronic immunosuppression, successful protocols will also require strategies that target potential effector cells.     

Cellular mechanisms underlying acute graft rejection: time for reassessment

Rejection of transplanted organs depends on an orchestrated immune response to histocompatibility antigens expressed by the grafted tissue. Effector mechanisms primarily responsible for the rejection process classically involve type 1 helper CD4(+) T cells, cytotoxic CD8(+) T cells and antibodies. Experimental studies revealed alternative mechanisms of rejection that implicate type 2 helper CD4(+) T cells and memory CD8(+) T cells as well as cells belonging to the innate immune system including natural killer cells, eosinophils and neutrophils. Furthermore, local inflammation associated with rejection is tightly regulated at the graft level by regulatory T cells and mast cells. The redundancy of rejection mechanisms explains the difficulty to induce transplantation tolerance and to develop reliable biomarkers for prediction of allograft outcome.     

The nature and mechanisms of DN regulatory T-cell mediated suppression

Regulatory T cells have been reported to enhance survival of transplanted allografts. We have recently identified and cloned a novel CD3(+)CD4(-)CD8(-) (double negative, DN) regulatory T cell from mice that were given a single class I mismatched donor lymphocyte infusion and permanently accepted donor-specific skin allografts. When infused into na?ve syngeneic mice, these DN T cells prolonged the survival of class I mismatched donor skin allografts. Here we further characterize the nature and mechanism of DN T-cell mediated suppression. This present study reveals that DN T cells are able to specifically eliminate activated syngeneic CD8(+) T cells that share the same T cell receptor (TCR) specificity as DN T cells in vitro. Similarly, we found that, along with an increase of recipient DN T cells in the peripheral blood, anti-donor CD8(+) T cells were also eliminated in vivo following a donor lymphocyte infusion. We further demonstrate that DN T regulatory cells do not mediate suppression by competition for growth factors or antigen presenting cells (APC) nor by modulation of APC, but require cell contact with the activated target CD8(+) T cells. This contact can be mediated either by the TCR on CD8(+) T cells that recognize constitutively expressed or acquired MHC molecules on DN T cells, or by the TCR on DN T cells that recognize constitutively expressed MHC molecules on CD8(+) T cells. Together, these data extend our previous findings, and expand the conditions in which DN T cells can potentially be used to specifically suppress allogeneic immune responses.     

Peripheral blood and intrarenal phagocytic chemiluminescence during acute kidney graft rejection

During organ graft rejection, soluble mediators of inflammation are released into the polymorphs (PMNs) and monocytes recruited from the blood. One functional capacity of polymorphs and monocytes/macrophages is the production of cytotoxic activated oxygen species upon stimulation, which may contribute to the rejection process. Nothing is known about the influence of allograft rejection on this inflammatory cell property. Chemiluminescence (CL) allows measurement of respiratory burst capacity in small cell samples. Zymosan-induced and luminol-amplified CL of diluted whole blood, separated PMNs, and mononuclear cells from peripheral venous blood, as well as of intragraft phagocytes was measured after allogeneic and autologous kidney transplantation in untreated dogs, CL of separated PMNs, mononuclear cells, and intragraft phagocytes was significantly elevated during allograft rejection. In autologous kidneys transplanted to recipients of allografts, CL was also increased in the autologous grafts during rejection of the allogeneic ones, indicating a systemic alteration in phagocyte function.     

Prolongation of allogeneic skin graft survival by injection of anti-Ly49A monoclonal antibody YE1/48

Ly49A receptors expressed on NK, NKT, and T cells play inhibitory roles in regulating the immune responses in vivo and in vitro. Whether or not injection of anti-Ly49A monoclonal antibody (mAb) YE1/48 can block allograft rejection has not been evaluated. Balb/c mouse recipients received intraperitoneal injections of YE1/48 mAb (0.5 mg) or control mAb or phosphate-buffered saline on days -1 and 10. On day 0, fully MHC-mismatched allogeneic C57BL/6 (B6) skin grafts were implanted. The skin graft survival and anti-donor humoral responses were observed. Whereas allogeneic B6 skin grafts survived 14 days in isotopy control antibody-treated or nontreated Balb/c mice, injection of YE1/48 mAb significantly prolonged B6 skin graft survival to 19 days (P < 0.0005). Injection of YE1/48 mAb into presensitized Balb/c recipients did not significantly delay B6 skin graft rejection. On the other hand, after depleting recipient NK, NKT, and some cytotoxic T cells by injection of anti-asialo GM1, YE1/48 failed to prolong B6 skin graft survival in Balb/c recipients. The present studies indicate that injection of YE1/48 mAb significantly delays allogeneic skin graft rejection in nonsensitized recipients but not in sensitized recipients. The presence of NK, NKT cells, and some cytotoxic T cells may be essential for YE1/48-mediated immunosuppression in vivo.     

Pluripotent allospecific CD8+ effector T cells traffic to lung in murine obliterative airway disease

Long-term success in lung transplantation is limited by obliterative bronchiolitis, whereas T cell effector mechanisms in this process remain incompletely understood. Using the mouse heterotopic allogeneic airway transplant model, we studied T cell effector responses during obliterative airways disease (OAD). Allospecific CD8+ IFN-gamma+ T cells were detected in airway allografts, with significant coexpression of TNF-alpha and granzyme B. Therefore, using IFN-gamma as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-gamma was produced by draining the lymph nodes, spleen, and lung mononuclear cells from allograft, but not isograft recipients by Day 14, and significantly decreased by Day 28. Although the majority of allospecific T cells were CD8+, allospecific CD4+ T cells were also detected in these compartments, with each employing distinct allorecognition pathways. An influx of pluripotent CD8+ effector cells with a memory phenotype were detected in the lung during OAD similar to those seen in the allografts and secondary lymphoid tissue. Antibody depletion of CD8+ T cells markedly reduced airway lumen obliteration and fibrosis at Day 28. Together, these data demonstrate that allospecific CD8+ effector T cells play an important role in OAD and traffic to the lung after heterotopic airway transplant, suggesting that the lung is an important immunologic site, and perhaps a reservoir, for effector cells during the rejection process.     

Intraparenchymal allografts in the mouse brain in relation to immunocytochemical identification of T lymphocyte subsets

In a study of intracerebellar allografts of mice brainstem anlagen (embryonic day 12-14), we examined immunocytochemically the expression of two different types of T lymphocytes in and around the grafts. Helper/inducer and cytotoxic/suppressor T cells were identified with anti-L3T4 and anti-Lyt-2 monoclonal antibodies, respectively. Allografts into major histocompatibility complex (MHC)-compatible recipients showed no histological signs of rejection such as marked neovascularization and cellular infiltrates even 6 months after transplantation, but those into MHC-incompatible recipients generally had rejection reactions within one month after transplantation. In the latter cases, the L3T4/Lyt-2 ratio for the T lymphocytes in the infiltrates of the grafts was 1.03 +/- 0.14 (mean +/- S.D.), suggesting that both helper/inducer and cytotoxic/suppressor T cells may play important roles in the mediation of intraparenchymal brain allograft rejection.     

The significance of timing of FTY720 administration on the immunosuppressive effect to prolong rat skin allograft survival

FTY720, a potent immunosuppressant, dramatically decreases the number of peripheral blood lymphocytes within a few hours after administration. The current study assessed the significance of timing of FTY720 administration on the immunosuppressive effect to prolong rat skin allograft survival (WKAH donor to F344 recipient). The median survival time of allografts was 7 days in the control recipients. FTY720 (1 mg/kg/day) significantly prolonged allograft survival when administered from days 0 and 3, but failed to exert an immunosuppressive effect when administered from day 4. Intragraft T cells, especially CD8(+) T cells, were markedly increased in number from day 4 to 6, peaking on day 5 in control recipients. FTY720 markedly decreased the number of intragraft CD8(+) T cells on day 5 when administered from days 0 and 3. In recipients administered with FTY720 from day 4, the number of intragraft CD8(+) T cells were only partially decreased on day 5. Intragraft CD8(+) T-cell number in those recipients on day 5 was almost the same as that in control recipients on day 4. In addition, FTY720 did not affect the increase in frequency of CD25(+) cells in the CD8(+) T-cell subset in allografts. It is likely that recipients treated with FTY720 from day 4 reject allografts by intragraft immune responses involved in CD8(+) T cells which had infiltrated before day 4, similar to control recipients. These findings suggest that FTY720 should be administered before increase in T cell infiltration into grafts to inhibit acute allograft rejection.     

Early T cell response to allografts occurring prior to alloantigen priming up-regulates innate-mediated inflammation and graft necrosis

The early inflammatory response within organ allografts is initiated by ischemia/reperfusion (I/R) and promotes subsequent alloantigen-primed T cell recruitment into and rejection of the graft. Polymorphonuclear leukocyte (PMN)-mediated tissue damage is a primary component of the early inflammation in allograft rejection. We sought to compare and elucidate the mechanism of early PMN infiltration into cardiac isografts and allografts. Despite identical production of PMN attractant chemokines, PMN infiltration following reperfusion into syngeneic and allogeneic grafts was not equivalent. PMN infiltration into isografts peaked at 9 to 12 hours post-transplant and quickly resolved. In contrast, PMN infiltration into allografts continued to elevated levels, peaking at 24 hours post-reperfusion. This amplified PMN infiltration into allografts did not resolve until 72 hours post-reperfusion and was accompanied by marked parenchymal necrosis. This early innate inflammatory response was regulated by IFN-gamma-producing CD8+ T cells present in the recipient before detectable alloantigen T cell priming. Co-culture with CD62L(low) CD8+ T cells, but not CD62L(high) CD8+ or CD62L(low) CD4+ T cells, harvested from na?ve animals induced allogeneic endothelial cells to express IFN-gamma-dependent chemokines. These data demonstrate CD8+ T cell-mediated attack on the vascular endothelium of allografts within hours following organ reperfusion that amplifies innate immune-mediated intra-graft inflammation and necrosis.     

Mode of alloantibody-mediated blockade of allo-sensitization for tumor allograft rejection

The mode of alloantibody-mediated inhibition of allo-sensitization for tumor allograft rejection was studied. Relatively small amounts of anti-H-2d alloantiserum administered shortly before or after injection of allogeneic spleen cells blocked the allo-sensitization for second-set tumor allograft rejection. In contrast, the alloantiserum injected shortly before inoculation of tumor barely enhanced the tumor growth. The passively administered alloantiserum inhibited the sensitization for allospecific cytotoxic T lymphocyte responses in vitro. Further study revealed that the allo-sensitization could be blocked with antiserum specific against only one of the expressed H-2 antigens on stimulator cells. Correspondingly, H-2Dd-monospecific monoclonal antibody (IgG2a) was effective in inhibiting the sensitization with cells expressing multiple H-2 alloantigens. These results suggest that antibody-mediated inactivation of stimulator cells as a whole is an important mechanism of the allograft enhancement.     

Blockade of the 4-1BB (CD137)/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival in mice

To explore the roles of 4-1BB (CD137) and CD28 in corneal transplantation, we examined the effect of 4-1BB/4-1BB ligand (4-1BBL) and/or CD28/CD80/CD86 blockade on corneal allograft survival in mice. Allogeneic corneal transplantation was performed between two strains of wild-type (WT) mice, BALB/c and C57BL/6 (B6), and between BALB/c and B6 WT donors and various gene knockout (KO) recipients. Some of the WT graft recipients were treated intraperitoneally with agonistic anti-4-1BB or blocking anti-4-1BBL monoclonal antibody (mAb) on days 0, 2, 4 and 6 after transplantation. Transplanted eyes were observed over a 13-week period. Allogeneic grafts in control WT B6 and BALB/c mice treated with rat immunoglobulin G showed median survival times (MST) of 12 and 14 days, respectively. Allogeneic grafts in B6 WT recipients treated with anti-4-1BB mAb showed accelerated rejection, with an MST of 8 days. In contrast, allogeneic grafts in BALB/c 4-1BB/CD28 KO and B6 CD80/CD86 KO recipients had significantly prolonged graft survival times (MST, 52.5 days and 36 days, respectively). Treatment of WT recipients with anti-4-1BB mAb resulted in enhanced cellular proliferation in the mixed lymphocyte reaction and increased the numbers of CD4(+) CD8(+) T cells, and macrophages in the grafts, which correlated with decreased graft survival time, whereas transplant recipients with costimulatory receptor deletion showed longer graft survival times. These results suggest that the absence of receptors for the 4-1BB/4-1BBL and/or CD28/CD80/CD86 costimulatory pathways promotes corneal allograft survival, whereas triggering 4-1BB with an agonistic mAb enhances the rejection of corneal allografts.     

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation.