Ruth Esser

Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers

Adoptive immunotherapy with allogeneic purified natural killer (NK) cell products might exert graft-versus-tumor alloreactivity with little risk of GVHD. In a prospective phase II study in two centers, we administered purified NK cell products to high-risk patients treated with haploidentical T-cell-depleted SCT. Sixteen patients received a total of 29 NK cell infusions on days +3, +40 and +100 after transplantation. Median doses (and ranges) of infused NK- and T-cells per product were 1.21 (0.3–3.8) × 107/kg and 0.03 (0.004–0.72) × 105/kg, respectively. With a median follow-up of 5.8 years 4/16 patients are alive. Cause of death was relapse in five, GVHD in three, graft failure in three, and transplant related neurotoxicity in one patient. Four patients developed acute GVHD⩾grade II, all receiving a total of ⩾0.5 × 105 T cells/kg. Compared with historical controls, NK cell infusions had no apparent effect on the rates of graft failure or relapse. Adoptive transfer of allogeneic NK cells is safe and feasible, but further studies are needed to determine the optimal dose and timing of NK cell therapy. Moreover, NK cell activation/expansion may be required to attain clinical benefit, while careful consideration must be given to the number of T cells infused.

IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA

NK group 2D (NKG2D)‐expressing NK cells exhibit cytolytic activity against various tumors after recognition of the cellular ligand MHC class I chain‐related gene A (MICA). However, release of soluble MICA (sMICA) compromises NKG2D‐dependent NK‐cell cytotoxicity leading to tumor escape from immunosurveillance. Although some molecular details of the NKG2D‐MICA interaction have been elucidated, its impact for donor NK (dNK) cell‐based therapy of solid tumors has not been studied. Within an ongoing phase I/II trial, we used allogeneic IL‐2 activated dNK cells after haploidentical stem cell transplantation for immunotherapy of patients with high‐risk stage IV neuroblastoma. NKG2D levels on activated dNK cells increased strongly when compared with freshly isolated dNK cells and correlated with enhanced NK‐cell cytotoxicity. Most importantly, elevated sMICA levels in patients plasma correlated significantly with impaired dNK‐cell‐mediated cytotoxicity. This effect could be reversed by high‐dose infusion of activated dNK cells, which display high levels of surface NKG2D. Our data suggest that the provided excess of NKG2D leads to clearance of sMICA and preserves cytotoxicity of dNK cells via non‐occupied NKG2D. In conclusion, our results identify this tumor immune escape mechanism as a target to improve immunotherapy of neuroblastoma and presumably other tumors.

Age-matched lymphocyte subpopulation reference values in childhood and adolescence: application of exponential regression analysis.

Background:u2002 Normal values of lymphocyte subpopulations for healthy children and adults have been published in defined age groups exclusively, which results in difficult data interpretation for patients close to the limit of contiguous age group ranges. In addition, normal values for a number of lymphocyte subpopulations have not been established to date.

Immune recovery in children undergoing allogeneic stem cell transplantation: absolute CD8+ CD3+ count reconstitution is associated with survival.

To evaluate the correlation between kinetics of immune reconstitution and survival, we prospectively evaluated lymphocyte subsets in 32 paediatric patients undergoing allogeneic stem cell transplantation (SCT) for haematological malignancies. Four-colour flow cytometric analysis was performed at short intervals with a median follow-up of 4 years post SCT. A total of 50% of patients reached age-matched 5th percentile of natural killer, cytotoxic T, B and helper T cells 4, 9, 20 and 28 weeks after SCT, respectively, which increased to more than 80% within 1 year after SCT. Transplantation of peripheral blood stem cells (PBSC) seemed to elicit the fastest reconstitution of CD3+, CD4+CD3+, CD8+CD3+ and naïve T cells compared to bone marrow (BM) or CD34-selected PBSC, which did not differ. Most importantly, we observed a significantly higher number of survivors among patients whose CD8+CD3+ absolute counts rose above the 5th percentile of age-matched normal levels during the first year post SCT compared to patients who never reached these levels (19/25 vs 0/7, P<0.001). This was still present in both subgroups, BM- and CD34-selected grafts (P=0.03, 0.02). These results from a small patient sample underline the importance of particular lymphocyte subsets for the outcome of children undergoing SCT. A larger study with detailed subset analysis is underway.

IL-2 Stimulated but Not Unstimulated NK Cells Induce Selective Disappearance of Peripheral Blood Cells: Concomitant Results to a Phase I/II Study

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipients immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipients peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9–14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/−) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients own CD69(−) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/−)CD69(+)NCR(high)CD62L(−) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.

Autologous transplantation of CD133 selected hematopoietic progenitor cells in a pediatric patient with relapsed leukemia

A pediatric patient with very early meningeal relapse of his CD34+ CD133− pre-B-ALL was transplanted with 2.5 × 106/kg CD133 selected autologous progenitor cells. Enrichment of CD133+ cells resulted in a purity of 92.3 ± 3.5% CD133+. Hematopoietic engraftment with >1.0 × 109/l neutrophils and >50 × 109/l platelets was reached within 13 and 24 days, respectively. At a follow-up of 11&frac12; months after autologous transplantation, the patient is in complete remission. To our knowledge, the successful transplantation with a CD133 selected graft is the first one to be reported worldwide. CD133 selected cells may serve as an alternative in the case of CD34+ malignancy.

Sequential anti-cytomegalovirus response monitoring may allow prediction of cytomegalovirus reactivation after allogeneic stem cell transplantation

Background Reconstitution of cytomegalovirus-specific CD3+CD8+ T cells (CMV-CTLs) after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary to bring cytomegalovirus (CMV) reactivation under control. However, the parameters determining protective CMV-CTL reconstitution remain unclear to date. Design and Methods In a prospective tri-center study, CMV-CTL reconstitution was analyzed in the peripheral blood from 278 patients during the year following HSCT using 7 commercially available tetrameric HLA-CMV epitope complexes. All patients included could be monitored with at least CMV-specific tetramer. Results CMV-CTL reconstitution was detected in 198 patients (71%) after allogeneic HSCT. Most importantly, reconstitution with 1 CMV-CTL per µl blood between day +50 and day +75 post-HSCT discriminated between patients with and without CMV reactivation in the R+/D+ patient group, independent of the CMV-epitope recognized. In addition, CMV-CTLs expanded more daramtaically in patients experiencing only one CMV-reactivation than those without or those with multiple CMV reactivations. Monitoring using at least 2 tetramers was possible in 63% (nu200a=u200a176) of the patients. The combinations of particular HLA molecules influenced the numbers of CMV-CTLs detected. The highest CMV-CTL count obtained for an individual tetramer also changed over time in 11% of these patients (nu200a=u200a19) resulting in higher levels of HLA-B*0801 (IE-1) recognizing CMV-CTLs in 14 patients. Conclusions Our results indicate that 1 CMV-CTL per µl blood between day +50 to +75 marks the beginning of an immune response against CMV in the R+/D+ group. Detection of CMV-CTL expansion thereafter indicates successful resolution of the CMV reactivation. Thus, sequential monitoring of CMV-CTL reconstitution can be used to predict patients at risk for recurrent CMV reactivation.

Multivariate analyses of immune reconstitution in children after allo-SCT: risk-estimation based on age-matched leukocyte sub-populations

The speed of immune recovery after allo-SCT is of central importance to overcome infectious complications and relapse. To evaluate the immune reconstitution of pediatric patients concerning overall survival, we developed a three-component multivariate model and generated a reference domain of ellipsoidal shape on the basis of normal leukocyte subtype values of 100 healthy children and adolescents. The leukocyte subtypes include absolute nos. of leukocytes, CD14+ monocytes, lymphocytes, CD3+ T cells, CD3+CD4+ helper T cells, CD3+CD8+ cytotoxic T cells, CD3−CD56+ natural killer-cells and CD19+ B cells, all of which are correlated, thus, requiring the application of multivariate as opposed to multiple univariate modeling. According to their immune reconstitution, 32 pediatric patients post allo-SCT were classified into low-risk and high-risk groups on the basis of our new model. Therefore, we evaluated if the patients reached the ellipsoid of normal leukocyte sub-population values post SCT. We detected a significantly higher number of long-time survivors among the low-risk group compared with the high-risk group at days 200 (P=0.001) and 300 (P<0.0001). This is superior to our previously published univariate analysis.1 Combined with the clinical observation, a classification into risk groups based on an extended patient cohort may represent a predictor for complications.

Hourly monitoring of circulating CD34+ cells to optimize timing of autologous apheresis in pediatric patients.

Summary:In order to increase the CD34+ cell yield in children undergoing autologous stem cell transplantation, the optimum time of apheresis after G-CSF administration has still to be found. We prospectively studied the mobilization of CD34+ cells and white blood cells in the peripheral blood (PB) of 20 pediatric patients before leukapheresis. The monitoring schedule covered 12u2009h, with blood samples taken before and at 2, 4, 5, 6, 7, 8, 10 and 12u2009h after G-CSF administration when 10 CD34+ cells/μl were reached. CD34+ cells were measured by flow cytometric analysis both in the single- and dual-platform setting. Two different patterns of mobilization (POM) emerged: 12 patients showed an increase in CD34+ cells in PB during the first 4u2009h after G-CSF (POM I), while eight patients had an initial decrease of CD34+ cells. However, all patients together showed a significant increase of CD34+ cells about 10u2009h after G-CSF administration. Further studies with more patients, using an enhanced monitoring schedule will be required to refine the results.