Belatacept Combined With Transient Calcineurin Inhibitor Therapy Prevents Rejection and Promotes Improved Long-Term Renal Allograft Function.

Belatacept, a T cell costimulation blocker, demonstrated superior renal function, lower cardiovascular risk, and improved graft and patient survival in renal transplant recipients. Despite the potential benefits, adoption of belatacept has been limited in part due to concerns regarding higher rates and grades of acute rejection in clinical trials. Since July 2011, we have utilized belatacept-based immunosuppression regimens in clinical practice. In this retrospective analysis of 745 patients undergoing renal transplantation at our center, we compared patients treated with belatacept (n = 535) with a historical cohort receiving a tacrolimus-based protocol (n = 205). Patient and graft survival were equivalent for all groups. An increased rate of acute rejection was observed in an initial cohort treated with a protocol similar to the low-intensity regimen from the BENEFIT trial versus the historical tacrolimus group (50.5% vs. 20.5%). The addition of a transient course of tacrolimus reduced rejection rates to acceptable levels (16%). Treatment with belatacept was associated with superior estimated GFR (belatacept 63.8 mL/min vs. tacrolimus 46.2 mL/min at 4 years, p < 0.0001). There were no differences in serious infections including rates of cytomegalovirus or BK viremia. We describe the development of a costimulatory blockade-based strategy that ultimately allows renal transplant recipients to achieve calcineurin inhibitor-free immunosuppression.

Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection.

The advent of costimulation blockade provides the prospect for targeted therapy with improved graft survival in transplant patients. Perhaps the most effective costimulation blockade in experimental models is the use of reagents to block the CD40/CD154 pathway. Unfortunately, successful clinical translation of anti-CD154 therapy has not been achieved. In an attempt to develop an agent that is as effective as previous CD154 blocking antibodies but lacks the risk of thromboembolism, we evaluated the efficacy and safety of a novel anti-human CD154 domain antibody (dAb, BMS-986004). The anti-CD154 dAb effectively blocked CD40-CD154 interactions but lacked crystallizable fragment (Fc) binding activity and resultant platelet activation. In a nonhuman primate kidney transplant model, anti-CD154 dAb was safe and efficacious, significantly prolonging allograft survival without evidence of thromboembolism (Median survival time 103 days). The combination of anti-CD154 dAb and conventional immunosuppression synergized to effectively control allograft rejection (Median survival time 397 days). Furthermore, anti-CD154 dAb treatment increased the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. This study demonstrates that the use of a novel anti-CD154 dAb that lacks Fc binding activity is safe without evidence of thromboembolism and is equally as potent as previous anti-CD154 agents at prolonging renal allograft survival in a nonhuman primate preclinical model.

Renal transplantation using belatacept without maintenance steroids or calcineurin inhibitors.

Kidney transplantation remains limited by toxicities of calcineurin inhibitors (CNIs) and steroids. Belatacept is a less toxic CNI alternative, but existing regimens rely on steroids and have higher rejection rates. Experimentally, donor bone marrow and sirolimus promote belatacept's efficacy. To investigate a belatacept-based regimen without CNIs or steroids, we transplanted recipients of live donor kidneys using alemtuzumab induction, monthly belatacept and daily sirolimus. Patients were randomized 1:1 to receive unfractionated donor bone marrow. After 1 year, patients were allowed to wean from sirolimus. Patients were followed clinically and with surveillance biopsies. Twenty patients were transplanted, all successfully. Mean creatinine (estimated GFR) was 1.10 ± 0.07 mg/dL (89 ± 3.56 mL/min) and 1.13 ± 0.07 mg/dL (and 88 ± 3.48 mL/min) at 12 and 36 months, respectively. Excellent results were achieved irrespective of bone marrow infusion. Ten patients elected oral immunosuppressant weaning, seven of whom were maintained rejection-free on monotherapy belatacept. Those failing to wean were successfully maintained on belatacept-based regimens supplemented by oral immunosuppression. Seven patients declined immunosuppressant weaning and three patients were denied weaning for associated medical conditions; all remained rejection-free. Belatacept and sirolimus effectively prevent kidney allograft rejection without CNIs or steroids when used following alemtuzumab induction. Selected, immunologically low-risk patients can be maintained solely on once monthly intravenous belatacept.

CTLA4Ig prevents alloantibody formation following nonhuman primate islet transplantation using the CD40-specific antibody 3A8.

Islet transplantation to treat type 1 diabetes has been limited in part by toxicities of current immunosuppression and recipient humoral sensitization. Blockade of the CD28/CD80/86 and CD40/CD154 pathways has shown promise to remedy both these limitations, but translation has been hampered by difficulties in translating CD154-directed therapies. Prior CD40-directed regimens have led to prolonged islet survival, but fail to prevent humoral allosensitization. We therefore evaluated the addition of CTLA4Ig to a CD40 blockade-based regimen in nonhuman primate (NHP) alloislet transplantation. Diabetic rhesus macaques were transplanted allogeneic islets using the CD40-specific antibody 3A8, basiliximab induction, and sirolimus with or without CTLA4Ig maintenance therapy. Allograft survival was determined by fasting blood glucose levels and flow cytometric techniques were used to test for donor-specific antibody (DSA) formation. CTLA4Ig plus 3A8, basiliximab and sirolimus was well tolerated and induced long-term islet allograft survival. The addition of CTLA4Ig prevented DSA formation, but did not facilitate withdrawal of the 3A8-based regimen. Thus, CTLA4Ig combines with a CD40-specific regimen to prevent DSA formation in NHPs, and offers a potentially translatable calcineurin inhibitor-free protocol inclusive of a single investigational agent for use in clinical islet transplantation without relying upon CD154 blockade.

Donor-directed MHC class I antibody is preferentially cleared from sensitized recipients of combined liver/kidney transplants.

For patients with chronic renal and liver diseases, simultaneous liver and kidney transplantation (SLKT) is the best therapeutic option. The role of a pretransplant donor-specific antibody (DSA) in SLKT is unclear. We report the results of a retrospective review from 7/08 to 10/09 of SLKT at our institution. Monitoring of DSA was performed using single antigen bead assay. Between 7/08 and 10/09, there were six SLKT who had preformed DSA and positive XM (four class I and II DSA, one class I DSA only, one class II only). One-year patient and renal graft survival was 83%. Death-censored liver allograft survival was 100%. Acute humoral rejection (AHR) of the kidney occurred in 66% (three with both class I and II DSA and one with only class II DSA) of patients. In those with AHR, class I antibodies were rapidly cleared (p < 0.01) while class II antibodies persisted (p = 0.25). All patients who had humoral rejection of their kidney had preformed anticlass II antibodies. Liver allografts may not be fully protective of the renal allograft, especially with pre-existing MHC class II DSA. Long-term and careful follow-up will be critical to determine the impact of DSA on both allografts.

Functional expression of the costimulatory molecule, B7/BB1, on murine dendritic cell populations.

Whereas dendritic cells (DC) are known to be potent activators of T cells both in vitro and in vivo, the critical costimulatory molecules expressed on DC are not well characterized. Using immunocytochemical and molecular techniques we find that splenic DC express B7/BB1, the counter-receptor for CD28. Moreover, expression of B7/BB1 is upregulated on epidermal Langerhans cells (LC) during their functional maturation into potent T cell stimulators. In blocking experiments, we find that participation of B7/BB1 is required for optimal proliferation of unprimed, allogeneic T cells in DC-driven, primary mixed leukocyte reactions. These data demonstrate that the regulated expression of B7/BB1 on DC may be important in the initiation of a primary T cell response.

4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses.

The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.

Experience with a novel efalizumab-based immunosuppressive regimen to facilitate single donor islet cell transplantation.

Islet transplantation is an experimental therapy for selected patients with type 1 diabetes (T1DM). It remains limited by immunosuppressive drug toxicity, progressive loss of insulin independence, allosensitization and the need for multiple islet donors. We describe our experience with an efalizumab-based immunosuppressive regimen as compared to the prevailing standard regimen, the Edmonton protocol. Twelve patients with T1DM received islet transplants: eight were treated with the Edmonton protocol; four were treated with daclizumab induction, a 6-month course of tacrolimus, and maintenance with efalizumab and mycophenolate mofetil. The primary endpoint was insulin independence after one islet infusion. Only two Edmonton protocol treated patients achieved the primary endpoint; six required islets from multiple donors, and all experienced leukopenia, mouth ulcers, anemia, diarrhea and hypertransaminasemia. Four became allosensitized. All patients treated with the efalizumab-based regimen achieved insulin independence with normal hemoglobin A1c after a single islet cell infusion and remained insulin independent while on efalizumab. These patients experienced significantly fewer side effects and none became allosensitized. Trial continuation was terminated by withdrawal of efalizumab from the market. These data suggest that this efalizumab-based regimen prevents islet rejection, is well tolerated, and allows for single donor islet transplantation.

Expanded nonhuman primate tregs exhibit a unique gene expression signature and potently downregulate alloimmune responses.

We have established two complementary strategies for purifying naturally occurring regulatory T cells (Tregs) from rhesus macaques in quantities that would be sufficient for use as an in vivo cellular therapeutic. The first strategy identified Tregs based on their being CD4+/CD25(bright). The second incorporated CD127, and purified Tregs based on their expression of CD4 and CD25 and their low expression of CD127. Using these purification strategies, we were able to purify as many as 1x10(6) Tregs from 120 cc of peripheral blood. Cultures of these cells with anti-CD3, anti-CD28 and IL-2 over 21 days yielded as much as a 450-fold expansion, ultimately producing as many as 4.7x10(8) Tregs. Expanded Treg cultures potently inhibited alloimmune proliferation as measured by a carboxyfluorescein succinimidyl ester- mixed lymphocyte reaction (CFSE-MLR) assay even at a 1:100 ratio with responder T cells. Furthermore, both responder-specific and third-party Tregs downregulated alloproliferation similarly. Both freshly isolated and cultured Tregs had gene expression signatures distinguishable from concurrently isolated bulk CD4+ T-cell populations, as measured by singleplex reverse transcriptase-polymerase chain reaction (RT-PCR) and gene array. Moreover, an overlapping yet distinct gene expression signature seen in freshly isolated compared to expanded Tregs identifies a subset of Treg genes likely to be functionally significant.

Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection.

Simultaneous blockade of the CD40 and CD28 costimulatory pathways is an effective treatment strategy to promote allograft acceptance but does not lead to indefinite allograft survival. The immune mechanisms responsible for costimulation-independent rejection are not defined. Here we have studied the rejection responses of murine C57BL/6 recipients, which we show to be relatively resistant to inhibition by combined CD40/CD28 blockade. We demonstrate that asialo GM1(+) CD8(+) cells play a critical role in this costimulation blockade-resistant rejection. These results provide new insights into the costimulatory requirements for T-cell subsets and demonstrate for the first time that combined blockade of the CD40 and CD28 pathways does not adequately inhibit CD8-mediated skin allograft rejection. Furthermore, we provide evidence that asialo GM1 is a potentially important therapeutic target for CD8-dependent immune responses.

The CD40 pathway in allograft rejection, acceptance, and tolerance.

The CD40 pathway plays a critical role at many levels of the sensitization and effector phases of allograft rejection. In addition to the important signaling role of this pathway for T cell help and effector function, recent evidence suggests that the CD40 pathway can directly co-stimulate T cells, independently of its effect on the B7-CD28 pathway.

Apparent polycythaemia.

Patients with apparent polycythaemia are characterised by a raised packed cell volume (PCV; males above 0.51, females above 0.48) but normal red cell mass (RCM; less than 25% greater than predicted). Prediction and interpretation of RCM and PV should be based on height and weight, since the use of body weight alone is misleading. Patients with PCV values up to 0.60 may have apparent polycythaemia but only 18% have a reduced PV (relative polycythaemia). Therefore, the most common cause of the raised PCV is a change in RCM and/or PV within their normal ranges. The clinical associations and possible causes for the RCM/PV changes include male sex, obesity, dehydration, diuretics, smoking, hypertension, alcohol, arterial oxygen desaturation, renal disease and increased catecholamine levels. Retrospective studies of patients with apparent polycythaemia and information from other groups of polycythaemic patients suggest an increased risk of vascular occlusion, although other factors, such as hypertension and smoking, are also involved. Proposed management includes modification of possible underlying causes and examination for risk factors for vascular occlusion. In patients with PCV levels chronically above 0.54 venesection should be used, but patients with PCV values below this level should only be venesected if they are considered to be at risk of vascular occlusion. The suggested target value for PCV for venesected patients is 0.45 or below.

The use of aspirin in polycythaemia vera and primary thrombocythaemia.

In polycythaemia vera (PV; polycythaemia rubra vera, primary proliferative polycythaemia) and primary thrombocythaemia (PT; essential thrombocythaemia), occlusive complications in the microvasculature and larger vessels are a significant cause of morbidity and mortality. Central to the pathogenesis of these complications are the quantitative and qualitative platelet changes present in these myeloproliferative disorders. Aspirin irreversibly inactivates cyclo-oxygenase in platelets. This leads to a reduced production of platelet thromboxane A2 which has vasoconstricting and platelet aggregatory properties. In haematologically normal individuals, aspirin has been shown to reduce thrombo-embolic complications in populations at risk of these events. In PV and PT, aspirin has been shown to specifically eliminate the micro-circulatory and vasomotor manifestations and there is some evidence of a reduction in larger vessel occlusion. Low-dose aspirin has been shown to substantially reduce the raised thromboxane A2 production of platelets in PV and PT patients. The incidence of haemorrhagic side-effects of aspirin are minimized by the use of low doses. Haemorrhagic events are particularly found in patients with platelet counts > 1000 x 10(9)/l and these events are enhanced by aspirin therapy in these patients. Aspirin should be used with caution in patients with dyspeptic symptoms or a history of peptic ulceration or bronchospasm. Precise PCV control (< 0.45) and cytoreduction (platelets < 400 x 10(9)/l) should be used in patients with PV to minimize the vascular occlusion risk but routine cytoreduction is proposed only for those at particular risk of vascular occlusion in PT. In the acute presentation of patients with vascular occlusion, cytoreduction and an aspirin dose of 300 mg a day is proposed, reducing to 75 mg a day with the control of symptoms and signs, while 75 mg a day may play a role as prophylactic therapy in the prevention of thrombosis. However, there are no prospective studies in PT to demonstrate the benefit/risk profile and to confirm these recommendations, while a randomized prospective placebo-controlled study of low-dose aspirin in PV has only recently been initiated.

The importance of growth hormone in the regulation of erythropoiesis, red cell mass, and plasma volume in adults with growth hormone deficiency.

Total body water (TBW) is reduced in adult GH deficiency (GHD) largely due to a reduction of extracellular water. It is unknown whether total blood volume (TBV) contributes to the reduced extracellular water in GHD. GH and insulin-like growth factor I (IGF-I) have been demonstrated to stimulate erythropoiesis in vitro, in animal models, and in growing children. Whether GH has a regulatory effect on red cell mass (RCM) in adults is not known. We analyzed body composition by bioelectrical impedance and used standard radionuclide dilution methods to measure RCM and plasma volume (PV) along with measuring full blood count, ferritin, vitamin B12, red cell folate, IGF-I, IGF-binding protein-3, and erythropoietin in 13 adult patients with GHD as part of a 3-month, double blind, placebo-controlled trial of GH (0.036 U/kg.day). TBW and lean body mass significantly increased by 2.5 +/- 0.53 kg (mean +/- SEM; P < 0.004) and 3.4 +/- 0.73 kg (P < 0.004), respectively, and fat mass significantly decreased by 2.4 +/- 0.32 kg (P < 0.001) in the GH-treated group. The baseline RCM of all patients with GHD was lower than the predicted normal values (1635 +/- 108 vs. 1850 +/- 104 mL; P < 0.002). GH significantly increased RCM, PV, and TBV by 183 +/- 43 (P < 0.006), 350 +/- 117 (P < 0.03), and 515 +/- 109 (P < 0.004) mL, respectively. The red cell count increased by 0.36 +/- 0.116 x 10(12)/L (P < 0.03) with a decrease in ferritin levels by 39.1 +/- 4.84 micrograms/L (P < 0.001) after GH treatment. Serum IGF-I and IGF-binding protein-3 concentrations increased by 3.0 +/- 0.43 (P < 0.001) and 1.3 +/- 0.15 (P < 0.001) SD, respectively, but the erythropoietin concentration was unchanged after GH treatment. No significant changes in body composition or blood volume were recorded in the placebo group. Significant positive correlations could be established between changes in TBW and TBV, lean body mass and TBV (r = 0.78; P < 0.04 and r = 0.77; P < 0.04, respectively), and a significant negative correlation existed between changes in fat mass and changes in TBV in the GH-treated group (r = -0.95; P < 0.02). We conclude that 1) erythropoiesis is impaired in GHD; 2) GH stimulates erythropoiesis in adult GHD; and 3) GH increases PV and TBV, which may contribute to the increased exercise performance seen in these patients.

Evaluation of diagnostic criteria in polycythemia vera.

There is no single diagnostic marker for the only known type of primary acquired erythrocytosis, polycythemia vera (PV). The Polycythemia Vera Study Group (PVSG) used a combination of major and minor diagnostic criteria. However, these guidelines have some limitations and in the presence of newer diagnostic tools, have been re-evaluated. The recommendations of the Radionuclide Panel of the International Council for Standardization Hematology based on surface area are recommended over red blood cell mass (RCM) mL/kg expressions. Absolute erythrocytosis can be assumed in males and females with packed cell volume (PCV) values greater than 0.60 and greater than 0.56, respectively. A satisfactory strategy of investigation for a secondary erythrocytosis must be used. Hypoxemia, as well as renal and hepatic pathology, must be excluded. In unexplained absolute erythrocytoses, pO(2)(50) values and serum erythropoietin (EPO) levels should be examined. The latter can be disappointing in the confirmation of a secondary erythrocytosis, but elevated values contraindicate a diagnosis of a primary erythrocytosis. Establishment of a clonal marrow population supports a diagnosis of PV. Thus an acquired karyotypic abnormality is a major criterion. Palpable splenomegaly remains an important diagnostic marker. Scanning techniques to demonstrate splenic enlargement should be used with caution. Allowance must be made for interobserver and intraobserver differences and variation in normal spleen size with age and size of the subject. Splenomegaly demonstrated in this way should be taken as a minor criterion. An increased neutrophil count (>10 x 10(9)/L and >12.5 x 10(9)/L in smokers) is readily measurable and should replace total white blood cell count. The error in measurement of neutrophil alkaline phosphatase (NAP) score is large, making it an unsuitable diagnostic criterion. Neutrophil and platelet counts (>400 x 10(9)/L) should be taken as separate minor criteria. Endogenous erythroid colonies (EEC) grown from the peripheral blood have been used as a marker of PV, but it is an expensive technique that is not standardized and not totally specific for PV. Low serum EPO values found in the majority of patients with PV should hold a linked minor criterion position with EEC. Expert opinions should be obtained if bone marrow histology is to be used in the diagnosis of PV, but histology holds an important role in confirming the diagnosis. Semin Hematol 38(suppl 2):21-24.

Polycythemia.

Plethora ('a morbid condition due to excess of red corpuscles') has been recognized since antiquity as a manifestation of disease. The use of leeches and blood letting, which was for centuries the mainstay of therapy in many clinical situations, reflected the importance then attached to having too much blood. Greater knowledge has achieved greater precision, so that measurement of packed cell volume (PCV) or hematocrit now defines polycythemia (> 0.51 in men and > 0.48 in women, when blood is sampled without venous occlusion). Clinical and laboratory investigations, mainly in the last 30 years, have enabled the subdivision of polycythemia into different subtypes of radically different etiology which will be described in this Chapter. The last decade, however, has seen the advancing edge of research in this field passing from clinical and routine laboratory testing to studies of cellular proliferation at the molecular level. Detailed understanding is, however, as always elusive, with each new finding revealing deeper layers which in turn need elucidation. The field of polycythemia is nevertheless moving slowly into the molecular era.

Donor-recipient microchimerism is not required for tolerance induction following recipient pretreatment with donor-specific transfusion and anti-CD4 antibody. Evidence of a clear role for short-term antigen persistence.

There is considerable current interest in the possibility that long-term graft acceptance in clinical solid-organ transplantation might be dependent upon the development of a microchimeric state between the donor and recipient. This possibility has been prompted by the observation that in some transplant patients cells of donor origin can be detected in peripheral sites such as the skin, and it has been proposed that these cells play an essential role in maintaining graft survival. The hypothesis that peripheral microchimerism is an absolute requirement for the long-term survival of solid-organ allografts was tested in a well-characterized model of transplantation tolerance in which adult recipient mice are pretreated 28 days before transplant with a single donor-specific transfusion under the cover of a depleting anti-CD4 antibody. Mice pretreated with this protocol accept donor-specific cardiac allografts (MST > 100 days) but reject those of a third party (MST 16.5 days). The protocol leads to operational tolerance in the long term in that donor-specific skin grafts show prolonged survival while those from a third party strain are rejected acutely. Since peripheral blood contains haematopoietic stem cells we speculated that the success of the anti-CD4/DST protocol might be dependent on the development of microchimerism. To address this possibility the DST was irradiated before administration under anti-CD4 antibody cover in order to prevent donor stem cells in the transfusion from establishing a microchimeric state in the recipient animals. Mice in this group rejected their grafts acutely (MST 12 days), suggesting indeed that stem cells might be very important in the success of this model. However, when the protocol was modified by giving three additional doses of irradiated whole blood to increase the possibility that recipient T cells would be engaged during antibody-induced immunocompromise, graft prolongation was restored (MST > 100 days). These results demonstrate that persistence of donor antigen at the time of anti-CD4 antibody treatment is critical for the induction of unresponsiveness in this model and show that microchimerism is not an absolute requirement for long-term graft survival.

Fas-mediated cytotoxicity. An immunoeffector or immunoregulatory pathway in T cell-mediated immune responses?

Fas/Fas ligand interactions serve as a signaling pathway for apoptosis (1-3), an important regulatory mechanism in the development and function of the immune system (4-9). Recent evidence that Fas-dependent apoptosis is also an important mode of T cell cytotoxicity (10-13) suggested that Fas might play a critical role in the effector phase of T-dependent immune responses, such as allograft rejection. We observed that Fas transcripts are constitutively expressed in syngeneic and allogeneic murine cardiac transplants, while Fas ligand (FasL) is up-regulated only in rejecting allografts. Surprisingly, the absence of an intact Fas/FasL pathway did not alter the tempo of allograft rejection, even CD4-dependent rejection. These results indicate that Fas/FasL interactions are not essential mediators of T cell-induced allograft damage. Rather, as suggested in other studies, the Fas pathway may be principally involved in the regulation of clonal expansion and subsequent contraction of T cell populations during immune responses.