Increased Pretransplant Frequency of CD28+ CD4+ TEM Predicts Belatacept-Resistant Rejection in Human Renal Transplant Recipients.

While most human T cells express the CD28 costimulatory molecule constitutively, it is well known that age, inflammation, and viral infection can drive the generation of CD28null T cells. In vitro studies have demonstrated that CD28null cell effector function is not impacted by the presence of the CD28 costimulation blocker belatacept. As such, a prevailing hypothesis suggests that CD28null cells may precipitate costimulation blockade-resistant rejection. However, CD28+ cells possess more proliferative and multifunctional capacity, factors that may increase their ability to successfully mediate rejection. Here, we performed a retrospective immunophenotypic analysis of adult renal transplant recipients who experienced acute rejection on belatacept treatment as compared to those who did not. Intriguingly, our findings suggest that patients possessing higher frequency of CD28+ CD4+ TEM prior to transplant were more likely to experience acute rejection following treatment with a belatacept-based immunosuppressive regimen. Mechanistically, CD28+ CD4+ TEM contained significantly more IL-2 producers. In contrast, CD28null CD4+ TEM isolated from stable belatacept-treated patients exhibited higher expression of the 2B4 coinhibitory molecule as compared to those isolated from patients who rejected. These data raise the possibility that pretransplant frequencies of CD28+ CD4+ TEM could be used as a biomarker to predict risk of rejection following treatment with belatacept.

Belatacept-Resistant Rejection Is Associated With CD28+ Memory CD8 T Cells.

Recently, newer therapies have been designed to more specifically target rejection in an effort to improve efficacy and limit unwanted toxicity. Belatacept, a CD28-CD80/86 specific reagent, is associated with superior patient survival and graft function compared with traditional therapy, but its adoption as a mainstay immunosuppressive therapy has been tempered by increased rejection rates. It is essential that the underlying mechanisms associated with this rejection be elucidated before belatacept is more widely used. To that end, we designed a study in a nonhuman primate kidney transplant model where animals were treated with either a belatacept- or a tacrolimus-based immunosuppressive regimen. Interestingly, we found that elevated pretransplant frequencies of CD28+ CD8+ TEMRA cells are associated with rejection on belatacept but not tacrolimus treatment. Further analysis showed that the CD28+ CD8+ TEMRA cells rapidly lose CD28 expression after transplant in those animals that go on to reject with the allograft infiltrate being predominantly CD28- . These data suggest that CD28+ memory T cells may be resistant to belatacept, capable of further differentiation including loss of CD28 expression while maintaining effector function. The unique signaling requirements of CD28+ memory T cells provide opportunities for the development of targeted therapies, which may synergize with belatacept to prevent costimulation-independent rejection.

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.

Reliability of transpulmonary pressure-time curve profile to identify tidal recruitment/hyperinflation in experimental unilateral pleural effusion.

The stress index (SI) is a parameter that characterizes the shape of the airway pressure-time profile (P/t). It indicates the slope progression of the curve, reflecting both lung and chest wall properties. The presence of pleural effusion alters the mechanical properties of the respiratory system decreasing transpulmonary pressure (Ptp). We investigated whether the SI computed using Ptp tracing would provide reliable insight into tidal recruitment/overdistention during the tidal cycle in the presence of unilateral effusion. Unilateral pleural effusion was simulated in anesthetized, mechanically ventilated pigs. Respiratory system mechanics and thoracic computed tomography (CT) were studied to assess P/t curve shape and changes in global lung aeration. SI derived from airway pressure (Paw) was compared with that calculated by Ptp under the same conditions. These results were themselves compared with quantitative CT analysis as a gold standard for tidal recruitment/hyperinflation. Despite marked changes in tidal recruitment, mean values of SI computed either from Paw or Ptp were remarkably insensitive to variations of PEEP or condition. After the instillation of effusion, SI indicates a preponderant over-distension effect, not detected by CT. After the increment in PEEP level, the extent of CT-determined tidal recruitment suggest a huge recruitment effect of PEEP as reflected by lung compliance. Both SI in this case were unaffected. We showed that the ability of SI to predict tidal recruitment and overdistension was significantly reduced in a model of altered chest wall-lung relationship, even if the parameter was computed from the Ptp curve profile.

The Knife's Edge of Tolerance: Inducing Stable Multilineage Mixed Chimerism but With a Significant Risk of CMV Reactivation and Disease in Rhesus Macaques.

Although stable mixed-hematopoietic chimerism induces robust immune tolerance to solid organ allografts in mice, the translation of this strategy to large animal models and to patients has been challenging. We have previously shown that in MHC-matched nonhuman primates (NHPs), a busulfan plus combined belatacept and anti-CD154-based regimen could induce long-lived myeloid chimerism, but without T cell chimerism. In that setting, donor chimerism was eventually rejected, and tolerance to skin allografts was not achieved. Here, we describe an adaptation of this strategy, with the addition of low-dose total body irradiation to our conditioning regimen. This strategy has successfully induced multilineage hematopoietic chimerism in MHC-matched transplants that was stable for as long as 24 months posttransplant, the entire length of analysis. High-level T cell chimerism was achieved and associated with significant donor-specific prolongation of skin graft acceptance. However, we also observed significant infectious toxicities, prominently including cytomegalovirus (CMV) reactivation and end-organ disease in the setting of functional defects in anti-CMV T cell immunity. These results underscore the significant benefits that multilineage chimerism-induction approaches may represent to transplant patients as well as the inherent risks, and they emphasize the precision with which a clinically successful regimen will need to be formulated and then validated in NHP models.

First-Year Waitlist Hospitalization and Subsequent Waitlist and Transplant Outcome.

Frailty is associated with inferior survival and increased resource requirements among kidney transplant candidates, but assessments are time-intensive and costly and require direct patient interaction. Waitlist hospitalization may be a proxy for patient fitness and could help those at risk of poor outcomes. We examined United States Renal Data System data from 51 111 adult end-stage renal disease patients with continuous Medicare coverage who were waitlisted for transplant from January 2000 to December 2011. Heavily admitted patients had higher subsequent resource requirements, increased waitlist mortality and decreased likelihood of transplant (death after listing: 1-7 days: hazard ratio [HR] 1.24, 95% confidence interval [CI] 1.20-1.28; 8-14 days: HR 1.49, 95% CI 1.42-1.56; ≥15 days: HR 2.07, 95% CI 1.99-2.15; vs. 0 days). Graft and recipient survival was inferior, with higher admissions, although survival benefit was preserved. A model including waitlist admissions alone performed better (C statistic 0.76, 95% CI 0.74-0.80) in predicting postlisting mortality than estimated posttransplant survival (C statistic 0.69, 95% CI 0.67-0.73). Although those with a heavy burden of admissions may still benefit from kidney transplant, less utility is derived from allografts placed in this population. Current kidney allocation policy, which is based in part on longevity matching, could be significantly improved by consideration of hospitalization records of transplant candidates.

Antibody-Mediated Rejection in Sensitized Nonhuman Primates: Modeling Human Biology.

We have established a model of sensitization in nonhuman primates and tested two immunosuppressive regimens. Animals underwent fully mismatched skin transplantation, and donor-specific antibody (DSA) response was monitored by flow cross-match. Sensitized animals subsequently underwent kidney transplantation from their skin donor. Immunosuppression included tacrolimus, mycophenolate, and methylprednisolone. Three animals received basiliximab induction; compared with nonsensitized animals, they showed a shorter mean survival time (4.7 ± 3.1 vs. 187 ± 88 days). Six animals were treated with T cell depletion (anti-CD4/CD8 mAbs), which prolonged survival (mean survival time 21.6 ± 19.0 days). All presensitized animals showed antibody-mediated rejection (AMR). In two of three basiliximab-injected animals, cellular rejection (ACR) was prominent. After T cell depletion, three of six monkeys experienced early acute rejection within 8 days with histological evidence of thrombotic microangiopathy and AMR. The remaining three monkeys survived 27-44 days, with mixed AMR and ACR. Most T cell-depleted animals experienced a rebound of DSA that correlated with deteriorating kidney function. We also found an increase in proliferating memory B cells (CD20(+) CD27(+) IgD(-) Ki67(+) ), lymph node follicular helper T cells (ICOS(+) PD-1(hi) CXCR5(+) CD4(+) ), and germinal center (GC) response. Depletion controlled cell-mediated rejection in sensitized nonhuman primates better than basiliximab, yet grafts were rejected with concomitant DSA rise. This model provides an opportunity to test novel desensitization strategies.

Belatacept: is there BENEFIT for liver transplantation too?

The results of the multicenter belatacept liver transplant trial disappoint with respect to safety and efficacy, and new approaches will be required before this agent plays a role in liver transplant immunosuppression. See article by Klintmalm et al on page 1817.

A blinded, randomized, controlled trial of three doses of high-dose insulin in poison-induced cardiogenic shock.

BACKGROUND: High dose insulin (HDI) has proven superior to glucagon and catecholamines in the treatment of poison-induced cardiogenic shock (PICS) in previous animal studies. Standard recommendations for dosing of insulin vary and the optimal dose of HDI in PICS has not been established. Our hypothesis was a dose of 10 U/kg/hr of HDI would be superior to 1 U/kg/hr with cardiac output (CO) as our primary outcome measure in pigs with propranolol-induced PICS. METHODS: This was a blinded, prospective, randomized trial with 4 arms consisting of 4 pigs in each arm. The arms were as follows: placebo (P), 1 U/kg/hr (HDI-1), 5 U/kg/hr (HDI-5), and 10 U/kg/hr (HDI-10). Cardiogenic shock was induced with a bolus of 0.5 mg/kg of propranolol followed by an infusion of 0.25 mg/kg/min until the point of toxicity, defined as 0.75 x (HR x MAP) was reached. At this point the propranolol infusion was decreased to 0.125 mg/kg/min and a 20 mL/kg bolus of normal saline (NS) was administered. The protocol was continued for 6 hours or until the animals died. RESULTS: 2 pigs died in the P arm, 1 pig died each in the HDI-1 and HDI-5 arms, and all pigs lived in the HDI-10 arm. There was a statistically significant difference in dose by time interaction on CO of 1.13 L/min over the 6 hr study period (p = < 0.001). There was also a statistically significant difference in dose by time interaction on MAP, HR, and systemic vascular resistance (SVR). No statistically significant difference was found between any of the arms regarding glucose utilization. CONCLUSION: HDI was statistically and clinically significantly superior to placebo in this propranolol model of PICS. Furthermore a dose response over time was found where CO increased corresponding to increases in doses of HDI.

A novel monoclonal antibody to CD40 prolongs islet allograft survival.

The importance of CD40/CD154 costimulatory pathway blockade in immunosuppression strategies is well-documented. Efforts are currently focused on monoclonal antibodies specific for CD40 because of thromboembolic complications associated with monoclonal antibodies directed towards CD154. Here we present the rational development and characterization of a novel antagonistic monoclonal antibody to CD40. Rhesus macaques were treated with the recombinant anti-CD40 mAb, 2C10, or vehicle before immunization with keyhole limpet hemocyanin (KLH). Treatment with 2C10 successfully inhibited T cell-dependent antibody responses to KLH without significant peripheral B cell depletion. Subsequently, MHC-mismatched macaques underwent intraportal allogeneic islet transplantation and received basiliximab and sirolimus with or without 2C10. Islet graft survival was significantly prolonged in recipients receiving 2C10 (graft survival time 304, 296, 265, 163 days) compared to recipients receiving basiliximab and sirolimus alone (graft survival time 8, 8, 10 days). The survival advantage conferred by treatment with 2C10 provides further evidence for the importance of blockade of the CD40/CD154 pathway in preventing alloimmune responses. 2C10 is a particularly attractive candidate for translation given its favorable clinical profile.

Diagnostic challenges in the evaluation of hepatic grafts from donors with HELLP syndrome: case report and review of the literature.

HELLP syndrome (hemolysis, elevated liver function tests, low platelets) is a rare complication of pregnancy that can result in severe complications such as hepatic infarction, subcapsular liver hematomas, and maternal brain death from cerebral hemorrhage. Recently, several investigators have described cases of successful transplantation using livers procured from donors who suffered brain death as a result of HELLP syndrome. However, this new class of marginal liver donors must be approached with caution. We report the case of a 28-year-old pregnant woman who suffered brain death due to HELLP syndrome and was subsequently evaluated for potential liver donation. Although her transaminitis and other liver function tests were markedly improving during the final days of her hospital course, her liver demonstrated segments of necrosis during attempted procurement, and the histology revealed extensive centrilobular necrosis. This case suggests that peak values of serum transaminases, as well as partial resolution of transaminitis, appear to have limited predictive ability in determining the suitability of the hepatic graft for transplantation. Thus, donors with HELLP syndrome should be approached with caution, even in the setting of laboratory values suggesting minimal or resolving hepatic injury. Furthermore, there should be an additional emphasis on obtaining and reviewing histology of the potential graft to determine its suitability for transplantation.

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.

Nitrogen leaching from Douglas-fir forests after urea fertilization.

Leaching of nitrogen (N) after forest fertilization has the potential to pollute ground and surface water. The purpose of this study was to quantify N leaching through the primary rooting zone of N-limited Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] forests the year after fertilization (224 kg N ha(-1) as urea) and to calculate changes in the N pools of the overstory trees, understory vegetation, and soil. At six sites on production forests in the Hood Canal watershed, Washington, tension lysimeters and estimates of the soil water flux were used to quantify the mobilization and leaching of NO(3)-N, NH(4)-N, and dissolved organic nitrogen below the observed rooting depth. Soil and vegetation samples were collected before fertilization and 1 and 6 mo after fertilization. In the year after fertilization, the total leaching beyond the primary rooting zone in excess of control plots was 4.2 kg N ha(-1) (p = 0.03), which was equal to 2% of the total N applied. The peak NO(3)-N concentration that leached beyond the rooting zone of fertilized plots was 0.2 mg NO(3)-N L(-1). Six months after fertilization, 26% of the applied N was accounted for in the overstory, and 27% was accounted for in the O+A horizon of the soil. The results of this study indicate that forest fertilization can lead to small N leaching fluxes out of the primary rooting zone during the first year after urea application.

Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression.

A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. Mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. Nevertheless, this chimerism-induction regimen induced amongst the longest-lived stem cell chimerism reported to date for non-human primates and thus represents a platform upon which to evaluate emerging tolerance-induction strategies.

Optimal phasic tracheal gas insufflation timing: an experimental and mathematical analysis.

OBJECTIVE: To investigate the modulation of CO2 clearance by changes in the duration of tracheal gas flow application during tracheal gas insufflation (TGI). DESIGN: Combination of bench studies using a commercial test lung and a commercially available intensive care ventilator and mathematical analysis using a clearance model derived from first principles. SETTING: University pulmonary research laboratory. PATIENTS: None. INTERVENTIONS: Experiments using TGI were performed on a test lung at two combinations of tidal volume and frequency. TGI was limited to part of the expiratory phase (the terminal 10-100% of expiration), and two different TGI catheter flow rates were studied. Permutations over a range of compliances, dead-space volumes, catheter flows, and TGI durations were collected. A mathematical model incorporating key ventilatory and TGI-related variables was developed to provide a first-principles theoretical foundation for interpreting the experimental results. MEASUREMENTS AND MAIN RESULTS: In the physical model, alveolar Pco2 attained a minimum value with TGI flow applied during the terminal 40-60% of the expiratory phase, a finding that was consistent over an almost eight-fold range of expiratory time constants. The mathematical model shows the same qualitative pattern as the experimental model, indicating that the observed behaviors are not an experimental artifact. CONCLUSION: The optimal duration of expiratory TGI flow application is stable over a wide range of impedance characteristics. Such stability suggests that near maximal effect of expiratory TGI could be obtained by applying TGI flow solely within the final 50% of the expiratory phase. Such uniform restriction of the application profile might both simplify technique implementation and decrease adverse consequences.

NK cells mediate costimulation blockade-resistant rejection of allogeneic stem cells during nonmyeloablative transplantation.

Although T-cell CD28/CD40 costimulation blockade represents a powerful mechanism to promote immune tolerance during murine allotransplantation, it has not yet been successfully translated to clinical transplantation. We determined the impact of natural killer (NK) cells on costimulation blockade-resistant rejection of donor bone marrow. We found that NK cells represent a potent barrier to engraftment: host NK depletion led to increased donor stem cell survival, increased mixed hematopoietic chimerism and to engraftment of low doses of donor marrow (1 x 10(8)/kg) that were otherwise rejected. To understand the mechanisms of NK alloreactivity, we employed an in vivo NK-specific cytotoxicity assay. We found that an increased proportion of target cells were killed between days 2 and 8 after cell transfer, and that NK killing of parental targets was inducible: NK cells preprimed with allotargets were more efficient at their elimination upon reexposure. Finally, both transplant and in vivo NK-killing models were used to determine the contribution of LFA-1 to NK alloreactivity. Blockade of LFA-1 led to decreased NK-mediated killing, and increased alloengraftment. These results identify NK alloreactivity as an integral component to costimulation blockade-resistant rejection, and suggest that its inhibition may represent an important target in the clinical translation of tolerance-induction transplantation.

Mathematical models for pressure controlled ventilation of oleic acid-injured pigs.

One-compartment, mathematical models for pressure controlled ventilation, incorporating volume dependent compliances, linear and nonlinear resistances, are constructed and compared with data obtained from healthy and (oleic acid) lung-injured pigs. Experimental data are used to find parameters in the mathematical models and were collected in two forms. Firstly, the P(e)-V curves for healthy and lung injured pigs were constructed; these data are used to compute compliance functions for each animal. Secondly, dynamic data from pressure controlled ventilation for a variety of applied pressures are used to estimate resistance parameters in the models. The models were then compared against the collected dynamic data. The best mathematical models are ones with compliance functions of the form C(V) = a + bV where a and b are constants obtained from the P(e)-V curves and the resistive pressures during inspiration change from a linear relation P(r) = RQ to a nonlinear relation P(r) = RQ(epsilon) where Q is the flow into the one-compartment lung and epsilon is a positive number. The form of the resistance terms in the mathematical models indicate the possible presence of gas-liquid foams in the experimental data.

Characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance.

Simultaneous blockade of the CD28 and CD40 T cell costimulatory pathways has been shown to effectively promote skin allograft survival in mice. Furthermore, blockade of one or both of these pathways has played a central role in the development of strategies to induce mixed hematopoietic chimerism and allospecific tolerance. It has recently been observed that the beneficial effects of CD40 blockade and donor splenocytes in prolonging skin graft survival can be abrogated by some viral infections, including lymphocytic choriomeningitis virus (LCMV). In this study, we show that LCMV infection prevents prolonged allograft survival following CD28/CD40 combined blockade. We further show that LCMV prevents the induction of allospecific tolerance and mixed hematopoietic chimerism, while delay of infection for 3-4 wk posttransplant has no effect on tolerance induction. Because of reports of anti-H-2(d) activity following LCMV infection, we assayed the ability of LCMV-specific T cells to respond to alloantigen at a single cell level. Although we confirm that LCMV infection induces the generation of alloreactive cells, we also demonstrate that LCMV-specific T cells do not divide in response to alloantigen. The alloresponse suppressed by costimulation blockade is restored by LCMV infection and correlates with increased dendritic cell maturation. We hypothesize that the costimulation blockade-resistant rejection mediated by LCMV could be partly attributable to the up-regulation of alternative costimulatory pathways subsequent to LCMV-induced dendritic cell maturation.