Debra D. Bloom

Campath-1H induction plus rapamycin monotherapy for renal transplantation: results of a pilot study.

Campath‐1H, an anti‐CD52 monoclonal antibody, was used as induction therapy (40 mg i.v. total dose) in 29 primary human renal transplants, and the patients were maintained on rapamycin monotherapy (levels 8–15 ng/mL) post‐transplant. Campath‐1H profoundly depletes lymphocytes long‐term and more transiently depletes B cells and monocytes. All patients are alive and well at 3–29 months of follow up. One graft was lost because of rejection. There have been no systemic infections and no malignancies. Eight of 29 patients have experienced rejection, which was successfully treated in seven of eight patients. Five of these patients had pathological evidence of a humoral component of their rejection. Seven of the 29 patients were converted to standard triple therapy on account of rejection. Rapamycin was generally well tolerated in that there were no significant wound‐healing problems; two lymphoceles required surgical drainage; and most patients were treated with a lipid‐lowering agent. Flow crossmatch testing post‐transplant revealed evidence of alloantibody in two patients tested with previous combined cellular and humoral rejection. Biopsies have shown no chronic allograft nephropathy to date. In view of the relatively high incidence of early humoral rejection, we plan to modify the immunosuppressive regimen in subsequent pilot studies. This clinical trial provides insight into the use of Campath‐1H induction in combination with rapamycin maintenance monotherapy.

CD4+CD25+FOXP3+ regulatory T cells increase de novo in kidney transplant patients after immunodepletion with campath-1H

Campath‐1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath‐1H‐treated renal allograft recipients for the presence of FOXP3+ regulatory T (Treg) cells. Flow cytometry demonstrated that CD4+CD25+FOXP3+ lymphocytes increased significantly within the CD4+ T‐cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti‐CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath‐1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath‐1H, with or without anti‐CD3, activation led to an increase in CD4+CD25+FOXP3+ cells that had suppressive capabilities. Together, these data suggest that Campath‐1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo.

BAFF is increased in renal transplant patients following treatment with alemtuzumab

Alemtuzumab is a monoclonal antibody that depletes T and B cells and is used as induction therapy for renal transplant recipients. Without long‐term calcineurin inhibitor (CNI) therapy, alemtuzumab‐treated patients have a propensity to develop alloantibody and may undergo antibody‐mediated rejection (AMR). In pursuit of a mechanistic explanation, we analyzed peripheral B cells and serum of these patients for BAFF (Blys) and BAFF‐R, factors known to be integral for B‐cell activation, survival, and homeostasis. Serum BAFF levels of 22/24 alemtuzumab‐treated patients were above normal range, with average levels of 1967 pg/mL compared to 775 pg/mL in healthy controls (p = 0.006). BAFF remained elevated 2 years posttransplant in 78% of these patients. BAFF‐R on CD19+ B cells was significantly downregulated, suggesting ligand/receptor engagement. BAFF mRNA expression was increased 2–7‐fold in CD14+ cells of depleted patients, possibly linking monocytes to the BAFF dysregulation. Addition of recombinant BAFF to mixed lymphocyte cultures increased B‐cell activation to alloantigen, as measured by CD25 and CD69 coexpression on CD19+ cells. Of note, addition of sirolimus (SRL) augmented BAFF‐enhanced B‐cell activation whereas CNIs blocked it. These data suggest associations between BAFF/BAFF‐R and AMR in alemtuzumab‐treated patients.

T-lymphocyte Alloresponses of Campath-1h-treated Kidney Transplant Patients

Background. Kidney transplant patients given Campath-1H (Alemtuzumab) immunodepletion therapy and long-term rapamycin monotherapy have excellent graft survival and function at three years. As an initial step in understanding the characteristics of repopulated T lymphocytes in these patients, we performed several assays to assess alloreactivity. Methods. We measured T-cell responses using CFSE-labeled recipient lymphocytes in a direct one-way MLR, and also analyzed the kinetics of expression of IFN-γ. We examined the T-cell responses of Campath-treated transplant patients on monotherapy versus those treated with anti-CD25 (Basiliximab) induction therapy and maintenance immunosuppression consisting of cyclosporine A, mycophenolate mofetil, and steroids. Results. On average, proliferative responses to donor antigen were equal between Campath and control groups. However, the Campath group displayed a greater response to third party compared to donor antigen (CD3+ P=0.04, CD4+ P=0.07, CD8+ P<0.01), whereas the control group did not display a greater response to third party (CD3+ P=0.69, CD4+ P=0.72, CD8+ P=0.60). Interestingly, more Campath patients (4 of 15) than control patients (0 of 8) displayed donor specific unresponsiveness as gauged by IFN-γ expression and T-cell proliferation (P=0.15). Conclusions. These studies suggest that Campath-1H in conjunction with rapamycin monotherapy retains intact immune responses to third party alloantigen, yet may promote hyporesponsiveness to donor antigen.

Calcineurin Inhibitor Withdrawal After Renal Transplantation with Alemtuzumab: Clinical Outcomes and Effect on T‐Regulatory Cells

To address the results of calcineurin inhibitor (CNI) withdrawal after alemtuzumab induction relative to CNI continuation, we performed a pilot randomized clinical trial in renal allograft recipients on CNI, a mycophenolic acid derivative and steroids after the first 2 months posttransplantation. Forty patients were randomized to taper off CNI or to maintain it, and followed for at least 1 year. Four patients in the withdrawal group were treated for acute rejection while no patient received antirejection treatment in the control group. Two control patients withdrew CNI due to nephrotoxicity. Estimated GFR was similar in both groups after 1 year. Flow cytometry of CD4+CD25+CTLA‐4+FoxP3+ regulatory T cells (Treg) demonstrated a significant increase in Treg percentages in the peripheral blood of alemtuzumab‐treated patients on CNI early postransplant. Furthermore, the increased Treg percentages in the withdrawal cohort were unchanged at month 6 postenrollment, whereas they decreased significantly in those patients maintained on CNI. Patients withdrawn from CNI after alemtuzumab trend toward a higher rejection rate, but most patients can be weaned from a CNI using this regimen. With the exception of maintaining increased Treg levels, the benefits are not appreciable in this short follow‐up, and a larger randomized trial is justified.

Monotherapy with the novel human anti-CD154 monoclonal antibody ABI793 in rhesus monkey renal transplantation model.

Background. This study assesses the safety and efficacy of the novel human anti-human CD154 monoclonal antibody ABI793 in rhesus monkeys. Methods. Outbred rhesus monkeys were used for renal transplantation from major histocompatibility complex-mismatched donors. Seven recipients were treated with ABI793, and six untreated recipients were used as controls. Graft function was monitored by urine output, serum creatinine, and renal biopsy. Phenotypic analysis of peripheral blood lymphocytes and mixed lymphocyte reaction were performed before transplantation and periodically after transplantation. Anti-donor major histocompatibility complex class I antibody levels were measured at the time of sacrifice. Results. Monkeys in the treated group demonstrated prolonged graft survival compared with controls. One monkey was sacrificed because of a urine leak on postoperative day 13. Three monkeys were sacrificed because of acute rejection (days 44, 149, and 158). Two monkeys were sacrificed because of chronic active rejection (days 154 and 221). One monkey was sacrificed on day 139 without rejection to observe the effects of ABI793 in the absence of rejection. There were no obvious clinical side effects of ABI793, but microscopic thromboembolic changes were observed in two monkeys. Lymphocyte subsets remained unaltered in all monkeys. Mixed lymphocyte reaction showed nonspecific suppression 6 weeks after transplantation. The monkeys with chronic active rejection showed relatively strong alloantibody responses. Conclusions. ABI793 induces prolonged renal allograft survival in rhesus monkeys. Nevertheless, thromboembolic complications may occur and chronic allograft nephropathy may develop after anti-CD154 treatment is discontinued.

Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System

BACKGROUND The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohns disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times. METHODS We evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials. RESULTS After only two passages, we were able to expand a mean of 6.6 × 10(8) MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10(8) cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. CONCLUSIONS The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.

Early and Limited Use of Tacrolimus to Avoid Rejection in an Alemtuzumab and Sirolimus Regimen for Kidney Transplantation: Clinical Results and Immune Monitoring

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27–39 months of follow‐up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed‐type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3–4 ng/mL. One patient showed clinical signs of rejection at month 9 post‐transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor‐specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid‐free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1‐year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

Correlation between human leukocyte antigen antibody production and serum creatinine in patients receiving sirolimus monotherapy after Campath-1H induction.

Background. In this study, we determined whether Campath-1H induction followed by sirolimus monotherapy inhibited alloantibody production in renal transplantation. Second, we evaluated the correlation between human leukocyte antigen (HLA) antibody production and serum creatinine levels. Methods. Sera were taken 1 to 24 months after transplantation from 24 patients treated with Campath-1H and sirolimus and tested for serum creatinine and HLA-specific antibody by using flow cytometry and enzyme-linked immunosorbent assay. Results. Ten (42%) of the 24 patients treated with Campath-1H and sirolimus produced HLA antibodies. Six of these 10 developed both donor-specific antibodies (DSAs) and non–donor-specific antibodies (NDSAs), whereas only NDSAs were detected in the other four patients. In patients with biopsy-diagnosed humoral rejection (C4d+), serum levels of both DSA and NDSA significantly correlated with patient serum creatinine levels. Rejection treatment successfully reduced both DSAs and NDSAs and reversed humoral rejection. Conclusions. The numeric relationship between serum creatinine and DSA levels suggests a causal relationship between alloantibody and transplant rejection.

Effect of chemical ablation of myenteric neurons on neurotransmitter levels in the rat jejunum

We have quantified neurotransmitter changes in the rat jejunum in which the myenteric neurons were ablated by serosal application of benzalkonium chloride. Within 2 days after benzalkonium chloride treatment, there was a 40% reduction in the activity of choline acetyltransferase, a specific marker for cholinergic neurons, and a 25% reduction in the amount of vasoactive intestinal peptide per centimeter length of jejunum. By 15 days, levels were comparable to those in control segments, and by 45 days after myenteric neuronal ablation, levels in treated tissues were twice those in controls. In contrast, we observed no reduction in the amount of leucine-enkephalin per centimeter length of jejunum at early times after benzalkonium chloride treatment, although by 45 days, levels of this neurotransmitter in treated segments of jejunum were more than twice those in controls. Significant increases in muscle thickness and tissue weight were also observed at 15, 30, and 45 days after myenteric neuronal ablation. Thus we have observed that in response to chemical ablation of myenteric neurons in the rat jejunum, there is a thickening of the smooth muscle layers and a compensatory increase in the production of certain neurotransmitters by the surviving neuronal elements in the gut.