Melanie Bremm

Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

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% (n = 176) 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 (n = 19) 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.

Advanced flowcytometric analysis of regulatory T cells: CD127 downregulation early post stem cell transplantation and altered Treg/CD3+CD4+-ratio in severe GvHD or relapse

Regulatory T cells (Tregs) are of crucial importance to suppress graft versus host disease (GvHD) post allogeneic stem cell transplantation (SCT), but are also known to impair antitumor immunity. However, Treg longitudinal studies are rare and in this respect advanced flowcytometric approaches for Treg characterization are necessary. To investigate the relation of both the percentage and the absolute numbers of Tregs on GvHD or relapse we measured CD4(+)CD25(+/hi)CD127(lo/-) Tregs in 239 peripheral blood (PB) samples of 16 patients during the first two years post-SCT. A 10-color flowcytometric panel was established to evaluate Treg subpopulations and has been tested in ten healthy individuals. In patients we demonstrated a decrease in CD127 expression on T cells early post-SCT which increases during the first year. Moreover, Tregs reached higher absolute numbers in patients with GvHD≤grade I compared to those with GvHD grades II-IV. In contrast, the percentage of Tregs was significantly higher in patients with GvHD grades II-IV or disease relapse compared to those without GvHD. These patients fit into the range of healthy individuals where a median value of 7.5% and 6.4% of T helper cells were characterized as CD4(+)CD25(+/hi)CD127(lo/-) and CD4(+)CD25(+/hi) Tregs, respectively. Furthermore, Tregs could be further subdivided into 40% naïve, 51% central memory and 9% effector memory Tregs. Our results showed for the first time a downregulation of CD127 expression on T cells including Tregs in patients early post-SCT. Additionally, new insights into the recovery of Tregs regarding GvHD and relapse were provided.

Age-matched dendritic cell subpopulations reference values in childhood.

Dendritic cells (DCs) are the most potent antigen‐presenting cells and are the key link between the innate and adaptive immune response. Only a few reports with study populations of up to 50 individuals have been published with age‐based reference values for DC subpopulations in healthy children. Therefore, we aimed to establish reference ranges in a larger study population of 100 healthy children, which allowed age‐matched subgroups. Most previous studies were performed using a dual‐platform approach. In this study, a single‐platform approach in a lyse no‐wash procedure was used. DC subpopulations were defined as follows: CD45+CD85k+HLA‐DR+CD14−CD16−CD33+ cells as myeloid DCs (mDCs) and CD45+CD85k+HLA‐DR+CD14−CD16−CD123+ cells as plasmacytoid DCs (pDCs). Reference ranges were established using a semi‐parametric regression of age‐matched absolute and relative DC counts. We found a significant decline with increasing age in the medians of mDCs (P = 0.0003) and pDCs per μl peripheral blood (PB) (P = 0.004) and in the 50%, 90% and 95% reference ranges. We also identified significantly lower absolute cell counts of mDCs per μl PB in girls than in boys for all age groups (P = 0.0015). Due to the larger paediatric study population and single‐platform approach, this study may give a more precise overview of the normal age‐matched development of DC subpopulations and may provide a basis for analyzing abnormal DC counts in different illnesses or therapies such as post stem cell transplantation.

Pre-Emptive Immunotherapy for Clearance of Molecular Disease in Childhood Acute Lymphoblastic Leukemia after Transplantation

Monitoring of minimal residual disease (MRD) or chimerism may help guide pre-emptive immunotherapy (IT) with a view to preventing relapse in childhood acute lymphoblastic leukemia (ALL) after transplantation. Patients with ALL who consecutively underwent transplantation in Frankfurt/Main, Germany between January 1, 2005 and July 1, 2014 were included in this retrospective study. Chimerism monitoring was performed in all, and MRD assessment was performed in 58 of 89 patients. IT was guided in 19 of 24 patients with mixed chimerism (MC) and MRD and by MRD only in another 4 patients with complete chimerism (CC). The 3-year probabilities of event-free survival (EFS) were .69 ± .06 for the cohort without IT and .69 ± .10 for IT patients. Incidences of relapse (CIR) and treatment-related mortality (CITRM) were equally distributed between both cohorts (without IT: 3-year CIR, .21 ± .05, 3-year CITRM, .10 ± .04; IT patients: 3-year CIR, .18 ± .09, 3-year CITRM .13 ± .07). Accordingly, 3-year EFS and 3-year CIR were similar in CC and MC patients with IT, whereas MC patients without IT experienced relapse. IT was neither associated with an enhanced immune recovery nor an increased risk for acute graft-versus-host disease. Relapse prevention by IT in patients at risk may lead to the same favorable outcome as found in CC and MRD-negative-patients. This underlines the importance of excellent MRD and chimerism monitoring after transplantation as the basis for IT to improve survival in childhood ALL.

Relation between Acute GVHD and NK Cell Subset Reconstitution Following Allogeneic Stem Cell Transplantation

One of the major challenges of allogeneic stem cell transplantation (allo-SCT) is to reduce the risk of graft-versus-host disease (GVHD) while boosting the graft-versus-leukemia (GVL) effect. The reconstitution of natural killer (NK) cells following allo-SCT is of notable interest due to their known capability to induce GVL without GVHD. Here, in this study, we investigate the association between the incidence and severity of acute graft-versus-host disease (aGVHD) and the early reconstitution of NK cell subsets following allo-SCT. We analyzed 342 samples from 107 patients using flow cytometry, with a focus on immature CD56high and mature cytotoxic CD56dim NK cells. Longitudinal analysis of immune reconstitution after allo-SCT showed that the incidence of aGVHD was associated with a delayed expansion of the entire NK cell population, in particular the CD56high subset. Notably, the disturbed reconstitution of the CD56high NK cells also correlated with the severity of aGVHD.

Development of three different NK cell subpopulations during immune reconstitution after pediatric allogeneic hematopoietic stem cell transplantation: prognostic markers in GvHD and viral infections

Natural killer (NK) cells play an important role following allogeneic hematopoietic stem cell transplantation (HSCT) exerting graft-versus-leukemia/tumor effect and mediating pathogen-specific immunity. Although NK cells are the first donor-derived lymphocytes reconstituting post-HSCT, their distribution of CD56++CD16− (CD56bright), CD56++CD16+ (CD56intermediate=int), and CD56+CD16++ (CD56dim) NK cells is explicitly divergent from healthy adults, but to some extent comparable to the NK cell development in early childhood. The proportion of CD56bright/CD56int/CD56dim changed from 15/8/78% in early childhood to 6/4/90% in adults, respectively. Within this study, we first compared the NK cell reconstitution post-HSCT to reference values of NK cell subpopulations of healthy children. Afterward, we investigated the reconstitution of NK cell subpopulations post-HSCT in correlation to acute graft versus host disease (aGvHD) and chronic graft versus host disease (cGvHD) as well as to viral infections. Interestingly, after a HSCT follow-up phase of 12 months, the distribution of NK cell subpopulations largely matched the 50th percentile of the reference range for healthy individuals. Patients suffering from aGvHD and cGvHD showed a delayed reconstitution of NK cells. Remarkably, within the first 2 months post-HSCT, patients suffering from aGvHD had significantly lower levels of CD56bright NK cells compared to patients without viral infection or without graft versus host disease (GvHD). Therefore, the amount of CD56bright NK cells might serve as an early prognostic factor for GvHD development. Furthermore, a prolonged and elevated peak in CD56int NK cells seemed to be characteristic for the chronification of GvHD. In context of viral infection, a slightly lower CD56 and CD16 receptor expression followed by a considerable reduction in the absolute CD56dim NK cell numbers combined with reoccurrence of CD56int NK cells was observed. Our results suggest that a precise analysis of the reconstitution of NK cell subpopulations post-HSCT might indicate the occurrence of undesired events post-HSCT such as severe aGvHD.

Treatment of relapse after allogeneic stem cell transplantation in children and adolescents with ALL: the Frankfurt experience

Therapy for post-transplant relapse of paediatric ALL is limited. Standardised curative approaches are not available. We hereby describe our local procedure in this life-threatening situation. A total of 101 ALL patients received their first allogeneic stem cell transplantation (SCT) in our institution. After relapse, our primary therapeutic goal was to cure the patient with high-dose chemotherapy or specific immunotherapy (HDCHT/SIT) followed by a second SCT from a haploidentical donor (transplant approach). If this was not feasible, low-dose chemotherapy and donor lymphocyte infusions (LDCHT+DLI) were offered (non-transplant approach). A total of 23 patients suffered a post-transplant relapse. Eight patients received HDCHT/SIT, followed by haploidentical SCT in 7/8. Ten received LDCHT+DLI. The eight patients treated with a second transplant and the ten treated with the non-transplant approach had a 4-year overall survival of 56% and 40%, respectively (P=0.232). Prerequisites for successful treatment of post-transplant relapse by either a second transplant or experimental non-transplant approaches are good clinical condition and the capacity to achieve haematological remission by the induction treatment element.

Cytotoxic potential of IL-15-activated cytokine-induced killer cells against human neuroblastoma cells.

Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite advances in therapy, the prognosis is poor and optimized therapies are urgently needed. Therefore, we investigated the antitumor potential of interleukin‐15 (IL‐15)‐activated cytokine‐induced killer (CIK) cells against different NB cell lines.

Optimization of individualized graft composition: CD3/CD19 depletion combined with CD34 selection for haploidentical transplantation

Excessive T‐cell depletion (TCD) is a prerequisite for graft manufacturing in haploidentical stem cell (SC) transplantation by using either CD34 selection or direct TCD such as CD3/CD19 depletion.