Anti-donor Responses Research Articles

Role of innate and acquired immune mechanisms in clinical intestinal transplant rejection.

Long-term outcomes of intestinal transplantation are limited by infection and rejection. To understand the underlying immune mechanisms, graft infiltrating and peripheral blood cells were analyzed using multiple ex vivo assays in intestinal transplantation recipients. Infiltrating cells from rejected (graft enterectomy for rejection) and accepted or quiescent (stoma closure in stable transplant recipients) grafts were isolated and phenotypically characterized as to subsets and Toll-like receptor expressions as well as functionally tested for antimicrobial and antidonor immune responses. Multiparameter antidonor immunity was also assessed serially in the peripheral blood. The graft infiltrating lymphocytes were mostly of recipient origin in all patients tested. In rejecting grafts, the predominant populations were TcRαβ(+)CD3(+)CD8(+) T cells, and CD14(+) monocytes that coexpressed Toll-like receptor-2, receptor-3, receptor-4, receptor-5, and receptor-9, suggesting innate immune activation. In quiescent allografts the major cell subsets were CD13(+)CD14(-) monocytes and CD4(+)CD25(+) T cells with possible regulatory functions. Infiltrating cells from rejected but not quiescent grafts proliferated in response to enteric bacterial and donor antigens as well as killed donor targets. Serial follow-up of peripheral blood indicated donor-specific posttransplant unresponsiveness in micro-cell-mediated lympholysis (m-CML) and mixed lymphocyte reaction (MLR) in recipients with quiescent grafts, but not in recipients with multiple rejection episodes. Enzyme-Linked ImmunoSpot assays yielded parallel results: granzyme-B with micro-cell-mediated lympholysis and interferon-γ with MLR tests. These results were consistent with the notion that rejection was associated with innate and acquired antimicrobial and antidonor immune reactivity and that patients with stable grafts were free from these deleterious effects.

Aquaporin 4 blockade is a novel strategy targeting ischemia/reperfusion injury in heart transplantation (TRAN1P.942)

Abstract Aquaporin 4 (AQP4) is the major water channel in the brain and its absence reduces brain edema in murine models of stroke.Recently, human and mouse cardiomyocytes were shown to express AQP4. Our goal was to test whether therapeutic inhibition of AQP4 reduces IRI and improves cardiac allografts survival. B6 mice were transplanted with BALB/c heart allografts subjected to 8 h of cold ischemia. Donors received AQP4 inhibitor 0.5h before harvest; hearts were perfused with and stored for 8 h on ice with the inhibitor. Recipients were given AQP4 inhibitor every 6h for 5 days. In the absence of additional immunosuppression, treatment with AQP4 inhibitor significantly prolonged heart allograft survival (MST 2d, n=24 vs. 6 d, n=5), decreased early T cell infiltration and the intragraft expression of IFNγ, granzyme B, perforin, FASL, CXCR3, ICOS, and CXCL10 in comparison to untreated controls. The frequencies of donor-reactive spleen cells secreting IFNγ in recipients treated with AQP4 inhibitor were also four fold lower than those in untreated controls. Blocking AQP4 during allograft storage and early after transplantation prolongs the survival of fully MHC-mismatched heart allografts subjected to 8 h CIS. The graft prolongation is associated with the reduced early memory T cell infiltration into the graft and decreased anti-donor responses. Our data identify AQP4 as a promising therapeutic target to diminish the detrimental effects of prolonged CIS on transplant outcome.

Haptoglobin enhances cardiac transplant rejection.

Early graft inflammation enhances both acute and chronic rejection of heart transplants, but it is unclear how this inflammation is initiated. To identify specific inflammatory modulators and determine their underlying molecular mechanisms after cardiac transplantation. We used a murine heterotopic cardiac transplant model to identify inflammatory modulators of early graft inflammation. Unbiased mass spectrometric analysis of cardiac tissue before and ≤72 hours after transplantation revealed that 22 proteins including haptoglobin, a known antioxidant, are significantly upregulated in our grafts. Through the use of haptoglobin-deficient mice, we show that 80% of haptoglobin-deficient recipients treated with perioperative administration of the costimulatory blocking agent CTLA4 immunoglobulin exhibited >100-day survival of full major histocompatibility complex mismatched allografts, whereas all similarly treated wild-type recipients rejected their transplants by 21 days after transplantation. We found that haptoglobin modifies the intra-allograft inflammatory milieu by enhancing levels of the inflammatory cytokine interleukin-6 and the chemokine MIP-2 (macrophage inflammatory protein 2) but impair levels of the immunosuppressive cytokine interleukin-10. Haptoglobin also enhances dendritic cell graft recruitment and augments antidonor T-cell responses. Moreover, we confirmed that the protein is present in human cardiac allograft specimens undergoing acute graft rejection. Our findings provide new insights into the mechanisms of inflammation after cardiac transplantation and suggest that, in contrast to its prior reported antioxidant function in vascular inflammation, haptoglobin is an enhancer of inflammation after cardiac transplantation. Haptoglobin may also be a key component in other sterile inflammatory conditions.

B Cell Activating Factor (BAFF) and a Proliferation Inducing Ligand (APRIL) Mediate CD40-Independent Help by Memory CD4 T Cells

Donor-reactive memory T cells undermine organ transplant survival and are poorly controlled by immunosuppression or costimulatory blockade. Memory CD4 T cells provide CD40-independent help for the generation of donor-reactive effector CD8 T cells and alloantibodies (alloAbs) that rapidly mediate allograft rejection. The goal of this study was to investigate the role of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in alloresponses driven by memory CD4 T cells. The short-term neutralization of BAFF alone or BAFF plus APRIL synergized with anti-CD154 mAb to prolong heart allograft survival in recipients containing donor-reactive memory CD4 T cells. The prolongation was associated with reduction in antidonor alloAb responses and with inhibited reactivation and helper functions of memory CD4 T cells. Additional depletion of CD8 T cells did not enhance the prolonged allograft survival suggesting that donor-reactive alloAbs mediate late graft rejection in these recipients. This is the first report that targeting the BAFF cytokine network inhibits both humoral and cellular immune responses induced by memory CD4 T cells. Our results suggest that reagents neutralizing BAFF and APRIL may be used to enhance the efficacy of CD40/CD154 costimulatory blockade and improve allograft survival in T cell-sensitized recipients.

Therapeutic blockade of LIGHT interaction with herpesvirus entry mediator and lymphotoxin β receptor attenuates in vivo cytotoxic allogeneic responses.

Tumor necrosis factor/tumor necrosis factor receptor superfamily members conform a group of molecular interaction pathways of essential relevance during the process of T-cell activation and differentiation toward effector cells and particularly for the maintenance phase of the immune response. Specific blockade of these interacting pathways, such as CD40-CD40L, contributes to modulate the deleterious outcome of allogeneic immune responses. We postulated that antagonizing the interaction of LIGHT expression on activated T cells with its receptors, herpesvirus entry mediator and lymphotoxin β receptor, may decrease T cell-mediated allogeneic responses. A flow cytometry competition assay was designed to identify anti-LIGHT monoclonal antibodies capable to prevent the interaction of mouse LIGHT with its receptors expressed on transfected cells. An antibody with the desired specificity was evaluated in a short-term in vivo allogeneic cytotoxic assay and tested for its ability to detect endogenous mouse LIGHT. We provide evidence for the first time that in mice, as previously described in humans, LIGHT protein is rapidly and transiently expressed after T-cell activation, and this expression was stronger on CD8 T cells than on CD4 T cells. Two anti-LIGHT antibodies prevented interactions of mouse LIGHT with its two known receptors, herpesvirus entry mediator and lymphotoxin β receptor. In vivo administration of anti-LIGHT antibody (clone 10F12) ameliorated host antidonor short-term cytotoxic response in wild type B6 mice, although to a lesser extent than that observed in LIGHT-deficient mice. The therapeutic targeting of LIGHT may contribute to achieve a better control of cytotoxic responses refractory to current immunosuppressive drugs in transplantation.

Harnessing alloreactivity to achieve anti-leukemic responses.

One of the hallmarks of a successful cancer is the development of one or more mechanisms to circumvent immune responses. Mechanisms by which acute myeloid leukemia circumvent immune responses that have been identified include loss of human leukocyte antigens, expression of ligands for inhibitory receptors, induction of the accumulation of suppressor cells, producing IDO and elimination of leukemia reactive cells.1 One possible approach to overcome these inhibitory hurdles is to harness alloreactive responses to achieve anti-leukemic responses. Explanations for the potent alloreactive responses have included the ability of the T-cell receptor (TCR) to crossreact with allogeneic major histocompatibility complex molecules presenting endogenous peptides or that alloreactive cells express two different TCR.2, 3 Initial attempts to harness alloreactivity have used allogeneic stem cell transplantation. In this procedure the recipient immune system is knocked down as part of the conditioning regimes, resulting in primarily donor anti-recipient responses following the stem cell transplant. While the donor anti-recipient response can generate graft-versus-leukemia responses, there is a high incidence of graft-versus-host disease (GVHD).4 Donor lymphocyte infusions following the development of tolerance have been used to enhance donor anti-tumor responses.5 In contrast, the cellular immunotherapy protocol, in which large numbers of haploidentical cells (1–2 × 108 CD3+ cells/kg) are infused in non-conditioned patients, results in both donor anti-recipient responses as well as recipient anti-donor responses.6 The recipient anti-donor responses are sufficient to eliminate the donor cells within several weeks, thereby preventing GVHD. Anti-leukemic responses were detected despite the elimination of the donor cells. This clinical trial has been reopened and ongoing studies are attempting to define the immune responses responsible for the anti-leukemic responses in this protocol.7 Possibilities to be tested include alloreactive cells crossreacting with leukemic cells, alloreactive responses reactivating memory T cells or the presence of activated CD3+ cells permitting anti-leukemic responses using non-TCR receptors. Preliminary results with the initial patients enrolled in the cellular immunotherapy trial have found increased expression of cytolytic effector molecules in recipient T cells, but little cytolytic activity based on low levels of CD107a expression. Rapid upregulation of PD-1 ligands on leukemic cells after infusion of donor cells was detected in 2/3 patients and could provide one explanation for the lack of response. Analysis of additional clinical trial patients combined with development of in vitro assays will help define how the use of alloreactivity can be improved to generate more potent anti-leukemic responses.

MSCs Isolated From Patients With Ischemic Vascular Disease Have Normal Angiogenic Potential

MSCs Isolated From Patients With Ischemic Vascular Disease Have Normal Angiogenic Potential

Combining autologous dendritic cell therapy with CD3 antibodies promotes regulatory T cells and permanent islet allograft acceptance.

Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation.

Donor Specific Regulatory T-Cell Expansion Is Associated With the Maintenance of Allograft Tolerance Induced By Transient Mixed Chimerism.

Background: We have previously reported successful induction of solid organ allograft tolerance in nonhuman primates (NHP) via a mixed chimerism approach. Since these recipients achieved tolerance after only transient hematopoietic chimerism, peripheral mechanisms are considered to be operative in the maintenance of tolerance. In the current study, we evaluated the role of donor specific regulatory T cells (Treg) in NHP that achieved long-term allograft survival. Method:Nine cynomolgus monkeys that achieved long-term (>360 days) allograft survival without maintenance immunosuppression [kidney (n=6), kidney/heart (n=2), and lung (n=1)] were investigated. Three recipients [heart alone (n=2) and kidney (n=1)] that received the same conditioning regimen but rejected their allografts after discontinuation of immunosuppression were evaluated as controls. Anti-donor and anti-third party responses in these recipients were evaluated by ELISPOT (INF-g) and expansion of donor specific Tregs was evaluated by MLR against donor and third party cells with CFSE/Foxp3 staining. Expanded T cells were sorted and their suppressive function was evaluated by inhibition of T cell activation by CD3/CD28 beads. Results: Donor-specific hyporesponsiveness was observed with ELISPOT in long-term survivors, but substantial anti-donor responses in recipients with rejection. (Fig.1) Foxp3+ cells proliferated significantly more after MLR with donor PBMC than when stimulated with self or third party PBMC in 5/7 long-term survivors. (Fig.2 representative flow cytometric analysis).Figure: No Caption available.Sorted Treg dominant cells expanded after MLR with donor cells showed suppressive effects in T cell activation. In contrast, there was no proliferation of FoxP3+ cells observed after MLR with donor cells in recipients with rejection. Conclusion: These data suggested that Tregs play a critical role in the maintenance of long-term allograft tolerance following induction of transient mixed chimerism.

High Pre-Transplant Frequencies of rATG Sensitive Anti-Donor Alloreactive Memory/Effector T-Cells Are Strongly Associated to Early Acute Cellular Rejection After Kidney Transplantation.

Memory/effector T cells play a major role in allograft rejection, however, clinical assessment of anti-donor cellular sensitization is not currently being evaluated in human transplantation. In this study, pre-transplant donor-specific memory/efector T-cell responses were retrospectively evaluated using the IFNγ Elispot assay, in a group of 150 consecutive kidney transplant recipients to assess the clinical impact of cellular sensitization before transplantation on allograft outcome. Most patients with high pre-transplant donor-specific cellular alloreactivity received allografts from cadaveric donors and showed increased HLA class I mismatches (p<0,05). No differences were observed between alloreactive and non alloreactive patients regarding type of end stage renal disease, immunosuppression (both maintenance and induction) or humoral sensitization (p=NS). In general, incidence of all types of biopsy proven acute rejection (BPAR) were not different between highly pre-transpalnt T-cell alloreactive patients and non alloreactive individuals (p=NS). When both antibody (ABM) and late (>8 weeks) T-cell mediated (TCM) BPAR were excluded, high pre-transplant frequencies of anti-donor T-cell responses predicted the risk of early TCM BPAR with high NPV (90,56%). In a multivariate analysis, presence of donor-specific cellular sensitization before transplantation was an independent variable associated with increased risk of early TCM BPAR (p=0,05). Importantly, pre-transplant anti-donor alloreactive patients treated with T-cell depleting agents (rATG) were protected from early TCM BPAR (p=0,009) In conclusion, monitoring donor-specific T-cell sensitization using a highly sensitive IFN-γ Elispot before transplantation might be useful to discriminate patients at increased risk of early TCM BPAR immediately after kidney transplantation and they may benefit of T-cell depleting agents as induction therapy.Figure: No Caption available.

B Cells and CD40/CD154 Interactions Are Required for CD8 T Cell Recovery Following Antibody-Mediated Lymphoablation.

Antibody-meditaed lymphoablation is commonly used as an induction therapy in transplant patients. However, recipients eventually reconstitute their T cell repertoire, develop anti-donor immune responses and reject transplanted organ. The goal of this study was to investigate the requirements for T cell recovery following lymphoablation in a mouse model of heterotopic cardiac transplantation. We previously reported that memory CD4 T cells are less susceptible to depletion by murine antithymocyte globulin (mATG) than CD8 T cells. Additional CD4 T cell depletion impaired CD8 T cell recovery after mATG treatment in B6 recipients of BALB/c heart allografts (0.2% vs 3-5% of spleen CD8 T cells recovered by d. 10 posttransplant) and in non-transplanted B6 mice. Analogous findings were made in recipients treated with anti-CD8 ± anti-CD4 mAbs demonstrating that the requirement for CD4 cell help is a common feature of antibody-mediated lymphoablation. In addition, memory CD8 T cell reconstitution depended on CD4 T cell help in non-transplanted B6 mice and in B6 isograft recipients treated with mATG. Treatment with blocking anti-CD154 mAb plus mATG inhibited memory CD8 T cell recovery in B6 heart allograft recipients similar to the levels observed after CD4 T cell depletion. Conversely, agonistic anti-CD40 mAb restored expansion of memory CD8 T cells following mATG treatment of CD4 T cell depleted heart allograft recipients. To test whether B cells are required for CD8 T cell recovery, we used B6.huCD20tg mice. Treatment with anti-human CD20 mAb depleted >90% of peripheral B cells and markedly inhibited CD8 T cell expansion in huCD20tg recipients compared to B cell-sufficient mice. Next, B cells were isolated from mATG treated recipients with or without CD4 depletion. In the absence of CD4 T cells, B cells expressed lower levels of MHC class I, CD80, CD86, and TNFa suggesting that both cognate interactions and cytokines promote CD8 T cell expansion. Our results show that CD4 T cell help mediated through B cells and CD40/CD154 interactions drives CD8 T cell reconstitution following antibody-mediated depletion. These findings may be used to improve the efficiency of lymphoablation in sensitized transplant recipients.

Posttransplant Inflammation Promotes Memory T Cell Recovery Following Antibody-Mediated Lymphoablation.

Antibody-mediated depletion is commonly used as an induction therapy in recipients of organ transplants subjected to prolonged cold ischemia. We used a murine model of heart transplantation to test how surgery-induced inflammation and ischemia/reperfusion injury affect the reconstitution of recipient T cell repertoire after lymphoablation. We previously reported that preexisting memory T cells rapidly expand and contribute to transplant rejection in B6 recipients of BALB/c heart allografts treated with murine antithymocyte globulin (mATG). Unexpectedly, memory CD8 T cell recovery was not influenced by specificity for donor alloantigens suggesting an important role for posttransplant inflammation. In support of this, CD8 T cells recovered faster in B6 isograft recipients than in non-transplanted mice treated with mATG. The increase in cold ischemic time from 0.5 h (typical in mouse heterotopic heart transplantation model) to 8 h (more closely resembling clinical situations) markedly enhanced the post-depletion expansion of memory CD8 T cells in allograft recipients (10 fold increase at d. 7 and 10 posttransplant). To inhibit local and systemic posttransplant inflammation, we administered neutralizing anti-TNFa Ab into B6 recipients of BALB/c heart allografts treated with mATG or control rIgG. TNFa neutralization decreased the numbers of donor-reactive IFNg producing cells in the spleens of mATG treated recipient by 60%, but had little effect on the anti-donor IFNg response in control treated mice. These results suggest that TNFa facilitates recovery of the T cell repertoire rather than priming of donor-reactive T cells in recipients treated with mATG. Our data demonstrate that inflammation arising from surgical trauma, ischemia/reperfusion injury and lymphoablation facilitates memory T cell reconstitution following antibody-mediated depletion. Therefore, neutralizing proinflammatory cytokines may be used to improve the efficacy of induction therapies in sensitized transplant recipients.

IL-21 Is a Key Regulator Coordinating Multiple Immune Responses in Chronic Allograft Rejection.

Aims: IL-21 is critical for developing chronic autoimmune and anti-viral responses. However, its role in cardiac allograft rejection is unknown. Our recent work has shown that IL-21 signaling is critical for maintaining T-cell survival under IL-2 deprivation conditions (in press) and in sustaining autoimmune responses to islets (in preparation). We, thus hypothesize that IL-21 is necessary in promoting chronic allo-immune responses during cardiac allograft rejection. Methods: We performed heart transplants in IL21-/-, BATF-/-, and wild-type B6 mice recipients with or without transient therapy with an IL-21 receptor fusion protein (IL-21R.Fc). The donors of heart allografts were either fully-mismatched Balb/c mice or class-II mismatched Bm12 mice. Results: We showed that donor Bm12 heart allografts were protected from chronic rejection in IL21-/- mice (>100 days) but not control B6 mice (56.3 ± 22.9 days) (n=10; p<0.001). In contrast, both IL-21-/- and B6 mice acutely rejected fully mismatched Balb/c cardiac allografts (<12 days; n=6; p=NS). The protection from chronic rejection in IL-21-/- recipients was confirmed with reductions in pathological (GAD) scores, intimal thickening [figure 1], and vascular infiltration (B-cells, T-cells & macrophages). The IL21-/- recipients also displayed reduced anti-donor responses as well as significantly lower IFN-g (but not IL-2) production by alloreactive T-cells. Importantly, the smooth muscle actin (SMAC) staining showed loss of SMAC in the arterial smooth muscle bed and abnormal accumulation in cardiomyocytes of wild-type recipients, whereas in IL21-/- mice the SMAC was intact. Additionally, the central role of IL-21 signaling in chronic rejection was validated in BATF-/- as well as IL-21R.Fc treated B6 recipients of Bm12 heart allografts, showing protection from chronic rejection (>100 days). Conclusions: We show a novel role for IL-21 signaling in promoting multiple immune responses during chronic allograft rejection. We propose that IL-21 is a key regulator of chronic allo-responses and contributes to endothelial cells and/or vascular SMC damage.Figure: No Caption available.

Posttransplant inflammation promotes memory T cell recovery following antibody-mediated lymphoablation (TRAN3P.900)

Abstract Antibody-mediated depletion is commonly used as an induction therapy in recipients of organ transplants subjected to prolonged cold ischemia. We tested how surgery-induced inflammation and ischemia/reperfusion injury affect the recipient T cell recovery after lymphoablation. We previously reported that preexisting memory T cells rapidly expand and mediate rejection in B6 recipients of BALB/c heart allografts treated with murine antithymocyte globulin (mATG). Memory CD8 T cell recovery was not influenced by specificity for donor alloantigens suggesting a role for posttransplant inflammation. In support of this, CD8 T cells recovered faster in B6 isograft recipients than in non-transplanted mice treated with mATG. The increase in cold ischemic time from 0.5 h to 8 h markedly enhanced the post-depletion expansion of memory CD8 T cells in allograft recipients (10 fold increase at d. 10 posttransplant). Treatment with neutralizing anti-TNFα Ab decreased the numbers of donor-reactive IFNγ producing cells in the spleens of mATG treated heart allograft recipient by 60%, but had little effect on the anti-donor responses in control IgG treated recipients suggesting that TNFα facilitates T cell recovery rather than priming. Our data show that posttransplant inflammation facilitates memory T cell reconstitution following antibody-mediated depletion and that neutralizing proinflammatory cytokines may improve the efficacy of induction therapies in sensitized transplant recipients.

An Unanticipated Role for Survivin in Organ Transplant Damage

Ischemia/reperfusion (I/R) injury is a major determinant of graft survival in kidney transplantation. Survivin, an inhibitor of apoptosis that participates in the control of mitosis and cell cycle progression, has been implicated in renal protection and repair after I/R injury; however, no study has been performed in the transplant setting. We investigated the role of survivin in modulating posttransplant I/R injury in syngeneic and allogeneic kidney grafts, and studied whether protection from I/R injury impacted on the recipient immune system, on chronic allograft nephropathy and rejection. We used genetically engineered mice with survivin haploinsufficiency and WT mice in which survivin over-expression was induced by gene-delivery. Survivin haploinsufficiency in syngeneic grafts was associated with exuberant I/R tissue injury, which triggered inflammation eventually resulting in graft loss. Conversely, survivin over-expression in the grafts minimized I/R injury and dysfunction in syngeneic grafts and in a clinically relevant fully MHC-mismatched allogeneic combination. In the latter, survivin over-expression translated into limited anti-donor adaptive immune response and less long-term allograft injury with protection from renal parenchymal damage. Our data support survivin over-expression in the graft as a novel target for protocols aimed at limiting tissue damage at the time of transplant ultimately modulating the recipient immune system.

Key Role for CD4 T Cells During Mixed Antibody-Mediated Rejection of Renal Allografts

We utilized mouse models to elucidate the immunologic mechanisms of functional graft loss during mixed antibody-mediated rejection of renal allografts (mixed AMR), in which humoral and cellular responses to the graft occur concomitantly. Although the majority of T cells in the graft at the time of rejection were CD8 T cells with only a minor population of CD4 T cells, depletion of CD4 but not CD8 cells prevented acute graft loss during mixed AMR. CD4 depletion eliminated antidonor alloantibodies and conferred protection from destruction of renal allografts. ELISPOT revealed that CD4 T effectors responded to donor alloantigens by both the direct and indirect pathways of allorecognition. In transfer studies, CD4 T effectors primed to donor alloantigens were highly effective at promoting acute graft dysfunction, and exhibited the attributes of effector T cells. Laser capture microdissection and confirmatory immunostaining studies revealed that CD4 T cells infiltrating the graft produced effector molecules with graft destructive potential. Bioluminescent imaging confirmed that CD4 T effectors traffic to the graft site in immune replete hosts. These data document that host CD4 T cells can promote acute dysfunction of renal allografts by directly mediating graft injury in addition to facilitating antidonor alloantibody responses.

Dendritic cells of myeloid lineage

Advances in surgery, patient management, and pharmacologic immunosuppression have reduced the incidence of acute allograft rejection. However, generation of therapies to promote donor-specific immunosuppression with minimal side-effects has proved to be a difficult task. To some extent, this is because of our limited knowledge on how Ag-presenting cells (APCs) like dendritic cells initiate and maintain the antidonor response in vivo. Herein, we link the classic concepts on the role of donor's dendritic cells as passenger leukocytes with the state-of-the-art findings in the field. Numerous studies are starting to unveil the plethora of mediators and interactions with leukocytes that trigger maturation of donor's dendritic cells in the grafts. The concept that donor's dendritic cells migrate from the grafts to secondary lymphoid organs to prime T cells has been challenged in murine models of lung or intestine transplantation, in which T cells can also be primed in the allograft. Increasing evidence suggests that recipient's dendritic cells present donor's intact major histocompatibility complex (MHC) molecules in lymphoid organs and that they infiltrate the grafts. A more complete understanding of the role of dendritic cells in allosensitization will help to develop better dendritic cell-based therapies to achieve the final goal of promoting donor-specific immunosuppression.

The protein kinase C inhibitor sotrastaurin allows regulatory T cell function

The novel immunosuppressant sotrastaurin is a selective inhibitor of protein kinase C isoforms that are critical in signalling pathways downstream of the T cell receptor. Sotrastaurin inhibits nuclear factor (NF)-κB, which directly promotes the transcription of forkhead box protein 3 (FoxP3), the key regulator for the development and function of regulatory T cells (Tregs). Our center participated in a randomized trial comparing sotrastaurin (n = 14) and the calcineurin inhibitor Neoral (n = 7) in renal transplant recipients. We conducted ex vivo mixed lymphocyte reaction (MLR) and flow cytometry studies on these patient samples, as well as in vitro studies on samples of blood bank volunteers (n = 38). Treg numbers remained stable after transplantation and correlated with higher trough levels of sotrastaurin (r = 0·68, P = 0·03). A dose-dependent effect of sotrastaurin on alloresponsiveness was observed: the half maximal inhibitory concentration (IC50 ) to inhibit alloactivated T cell proliferation was 45 ng/ml (90 nM). In contrast, Treg function was not affected by sotrastaurin: in the presence of in vitro-added sotrastaurin (50 ng/ml) Tregs suppressed the proliferation of alloactivated T effector cells at a 1:5 ratio by 35 versus 47% in the absence of the drug (P = 0·33). Signal transducer and activator of transcription 5 (STAT)-5 phosphorylation in Tregs remained intact after incubation with sotrastaurin. This potent Treg function was also found in cells of patients treated with sotrastaurin: Tregs inhibited the anti-donor response in MLR by 67% at month 6, which was comparable to pretransplantation (82%). Sotrastaurin is a potent inhibitor of alloreactivity in vitro, while it did not affect Treg function in patients after kidney transplantation.

Cellular Immunotherapy: Using Alloreactivity to Induce Anti-Leukemic Responses without Prolonged Persistence of Donor Cells

A goal of cancer immunologists is to harness cellular immune responses to achieve anti-cancer responses. One of the strongest activating stimuli for the immune system is the encounter with cells expressing allogeneic HLA molecules. While alloreactive responses can negatively impact of the outcome of hematopoietic stem cell transplant because of graft-versus-host disease (GVHD), these same responses can have anti-leukemic effects. Donor lymphocyte infusions have been used in an attempt to harness alloreactive responses to achieve anti-leukemic responses. Because this protocol is usually carried out in the absence of recipient anti-donor responses, this protocol often induces GVHD as well as anti-leukemic responses. A recent study indicated the infusion of large number of haploidentical donor cells (1–2 × 108 CD3+ cells/kg) into patients with refractory hematological malignancies (100 cGy total body irradiation) resulted in 14 (7 major) responses/26 patients. A rapidly developing cytokine storm was observed, while no persisting donor cells could be detected at two weeks after infusion eliminating the possibility of GVHD. Characterization of the effector mechanisms responsible for the anti-leukemic responses in this protocol, should guide new approaches for achieving enhanced anti-leukemic responses using this protocol.

ASKP1240, a Fully Human Anti-CD40 Monoclonal Antibody, Prolongs Pancreatic Islet Allograft Survival in Nonhuman Primates

A strategy for inhibiting CD40 has been considered as an alternative approach for immunosuppression because of undesirable effects of anti-CD154 monoclonal antibodies (mAbs). Previously, we demonstrated that ASKP1240, which is a fully human anti-CD40 mAb, significantly prolonged kidney and liver allograft survival in cynomolgus monkeys without causing thromboembolic complications. Herein, we evaluated the effect of ASKP1240 on pancreatic islet transplantation (PITx) in cynomolgus monkeys. Diabetes was induced by total pancreatectomy, and islet allografts were transplanted into the liver. Following PITx (8201–12 438 IEQ/kg), blood glucose levels normalized promptly in all animals. Control islet allografts were rejected within 9 days (n = 3), whereas ASKP1240 (10 mg/kg) given on postoperative days 0, 4, 7, 11 and 14 (induction treatment, n = 5) significantly prolonged graft survival time (GST) to >15, >23, 210, 250 and >608 days, respectively. When ASKP1240 (5 mg/kg) was administered weekly thereafter up to post-PITx 6 months (maintenance treatment, n = 4), GST was markedly prolonged to >96, >115, 523 and >607 days. During the ASKP1240 treatment period, both anti-donor cellular responses and development of anti-donor antibodies were abolished, and no serious adverse events were noted. ASKP1240 appears to be a promising candidate for immunosuppression in clinical PITx.