ALBERT

Description: Introduction: Acute leukemia is the most common malignancy in children and develops within the bone marrow. Consequently, bone marrow derived T cells of leukemia patients can be defined as tumor infiltrating lymphocytes (TILs). Dysfunctional TILs have been described in several other malignancies. However, in pediatric patients the interaction between leukemic blasts and TILs remains largely unknown. In order to understand the impact of leukemic blasts on bone marrow T cells we profiled T cells in the bone marrow of pediatric leukemia patients by surface marker and transcriptome wide analysis. Methods: First, artificial changes in marker expression due to cryopreservation and thawing were ruled out (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (HD, n=23) were identified in a local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T cells in primary (n=49) and relapse leukemia samples (n=28). As we could not detect surface CTLA4 expression on T cells, CTLA4 was stained intracellularly. Additionally, RNA-Seq on sorted bone marrow derived CD8 T cells (n=48; TCP-ALL: 12, AML: 20, HD: 16) was performed. Analysis of RNA-Seq data was based on Reads Per Kilobase Million (RPKM) normalization and False Discovery Rate (FDR, Benjamini-Hochberg) statistics. 172 differentially expressed genes were found when comparing bone marrow derived CD8 T cells from healthy donors (n=16) and leukemia patients (n=32) using the following criteria: RPKM〉2 in both groups, fold change〉2 and FDR

Description: Abstract 4542 Immunosuppressive treatment is widely used, especially after allogeneic stem cell transplantation (HSCT) to prevent or treat graft versus host disease (GvHD). Common drugs are Ciclosporin A or Tacrolimus in combination with steroids. However, immunosuppressive treatment and the underlying conditions are associated with an increased risk of viral reactivations with persistent pathogens like cytomegalovirus or adenovirus. In the absence of a protective immune response, virus infection remains a life-threatening complication after HSCT. Here we investigated the antiviral T-cell response (n=12) after ex vivo exposition with Ciclosporin A, Tacrolimus, Prednisolone or Mycophenolate at time of immunosuppression, 24 hours and 72 hours later. Analysis has been done with IFNgamma Elispot assays, confirmed by intracellular cytokine staining in flow cytometry and analysis of T-cell proliferation detected by CFSE. The antiviral T-cell response is suppressed after 24 hours using normal serum concentrations (100-200ng/ml) of cyclosporine A. T-cell annergy, induced by cyclosporine, could be reversible, after 72 without immunosuppression. Tacrolimus has a stronger immunosuppressive effect on T-cell activation within the same time and even low levels of 1ng/ml induce T-cell suppression after 72 hours. Peak levels of calcineurin inhibitors even suppressed the T-cell response to superantigens like staphylococcal antigen B or PHA. As expected, Prednisolone had a short and dose dependend effect on T-cell activation. Mycophenolate has a mild effect on the activation of virus-specific T-cells. However, all three drugs induced a significant reduction in Ag-specific T-cell proliferation. In conclusion, Interferonγ detection in virus-specific T-cells is a good diagnostic tool for clinicians to monitor the risk of viral complications in immunosuppressed patients. Tacrolimus, Cyclosporin A, Prednisolone and Mycophenolate induce an activation defect in Ag-specific T-cells with decreasing severity. The effect is reversible and corresponds to high or low serum levels. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Pediatric acute lymphoblastic leukemia (ALL) is a cancer entity of minimal mutational load and low immunogenicity. The interaction of ALL cells with bone marrow (BM) T cells has not been investigated as a pathogenic driver or prognostic marker for pediatric ALL. We defined BM T cells of pediatric ALL patients as tumor-infiltrating lymphocytes (TILs) and investigated the prognostic relevance of co-stimulatory and co-inhibitory signals between ALL and BM T cells. Methods BM samples of 100 pediatric ALL patients were analyzed at time of initial diagnosis. T-cell subpopulations and expression of co-stimulatory and co-inhibitory molecules were defined by flow cytometry and correlated with clinical outcome of the patients. To investigate the role of TIM-3 for the interaction between T cells and leukemic cells, CRISPR/Cas9-mediated TIM-3 knockout (KO) was performed in primary T cells by ribonucleoprotein electroporation. T-cell activation and proliferation after contact with leukemic target cells were analyzed in TIM-3 KO cells and compared to wildtype T cells and T cells with retroviral TIM-3 overexpression. Interaction of T cells with leukemic target cells was induced by addition of anti-CD19/-CD3 bispecific T-cell engager (BiTE). Fold change (FC) of T-cell activation and proliferation was analyzed before and after co-culture. BM expression levels of known TIM-3 inducers were identified by RNA next generation sequencing of the bone marrow samples. Results Multivariate analyses identified high TIM-3 expression on CD4+ BM T cells at initial diagnosis as strong predictor for relapse of pediatric acute lymphoblastic leukemia (relapse free survival (RFS) 94.6% vs. 70.3%). The risk to develop ALL relapse was 7.1-fold higher in the group of TIM-3 high expressing patients (n=37) compared to TIM-3 low expressing patients (n=37). Expression levels of known TIM-3 ligands and inducers in the bone marrow of the patients were analyzed by RNA next generation sequencing and compared between patients with high TIM-3 expression (n=12) and low TIM-3 expression (n=15) on BM T cells. Presence of known TIM-3 ligands HMGB1 (High-Mobility-Group-Protein B1) and Galectin-9 was confirmed, but expression levels did not show significant differences. Known TIM-3 inducers IL-2, -7, -15 and -21 were not expressed on RNA level indicating that another mechanism must be responsible for TIM-3 overexpression. In vitro experiments showed that the interaction with leukemic cells induces TIM-3 expression on the surface of T cells (mean TIM-3 expression 51.1% vs. 29.7% on T cells with vs. without addition of leukemic cells, n=3). To investigate the functional relevance of TIM-3 expression in pediatric leukemia, TIM-3 KO and overexpression was performed on primary T cells. TIM-3 KO T cells showed higher activation levels after co-culture with leukemic cell lines plus CD3-/CD19-specific BiTE compared to wildtype (WT) T cells (FC of CD69 surface expression 5.0 vs. 3.2, n=3). FC of anti-leukemic proliferation was impaired in TIM-3 overexpressing T cells compared to WT T cells (FC 1.6 vs. 2.3, n=3) whereas TIM-3 KO T cells showed a higher proliferation FC compared to controls (FC 6.5 vs. 2.4, n=3). Conclusions Our study identifies TIM-3 expression on CD4+ bone marrow T cells at initial diagnosis as a strong predictor for pediatric ALL relapse. TIM-3 expression is induced by interaction of T cells with leukemic cells and results in impaired anti-leukemic T-cell activation and proliferation. TIM-3-mediated T-cell inhibition represents a new mechanism of impaired immune surveillance in pediatric ALL and blockade of this axis may be of importance for future immunotherapy in ALL. Disclosures No relevant conflicts of interest to declare.

Description: Following allogeneic hematopoietic stem cell transplantation (alloHSCT), children are at risk of life-threatening pneumococcal infections. Whereas vaccination with polysaccharide vaccines fails to elicit protective immunity in most alloHSC transplant recipients, pneumococcal conjugate vaccines may effectively prevent invasive disease by eliciting T-cell–dependent antibody responses. Here, we report safety and immunogenicity in 53 children immunized with a regimen of 3 consecutive doses of a heptavalent pneumococcal conjugate vaccine (7vPCV) in monthly intervals starting 6 to 9 months after alloHSCT. Immunization was well tolerated with no vaccine-related serious adverse events. Serologic response rates evaluable in 43 patients ranged from 41.9% to 86.0% and 58.1% to 93.0% after 2 and 3 vaccinations, respectively, with 55.8% and 74.4% of patients achieving protective antibody levels to all 7 vaccine serotypes. Our study provides the first evidence that vaccination with 7vPCV is safe and elicits protective antipneumococcal antibody responses in pediatric recipients of related or unrelated donor alloHSC transplants within the first year following transplantation. This trial was registered at www.clinicaltrials.gov as NCT00169728.

Description: Key Points Adoptive transfer of TH-1 cells is a safe and effective treatment of refractory AdV infection after stem cell transplantation. AdV-related mortality was 9.5% in patients with a response to ACT (overall survival 71%) compared with 100% mortality in nonresponders.

Description: Relapsed and refractory B-precursor acute lymphoblastic leukemia (B-ALL) remains a major therapeutic problem. Chimeric antigen receptor (CAR) modified T cells targeting CD19 are promising treatment options for these patients with the potential to induce hematological remission in adult and pediatric patients with refractory B-ALL. Despite the promising data, some patients do not respond to T-cell treatment. Until now it is not possible to fully understand and predict critical factors for response or non-response, but proliferation and persistence of CAR T cells in vivo is an essential precondition for treatment efficacy. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to be the best candidates for a sustained in vivo expansion after T-cell therapy with small cell doses. Therefore, we set up a protocol for the generation of anti-CD19 CAR T cells in a closed system that is compliant to current GMP regulations. Starting samples were mononuclear cells from pediatric ALL patients at diagnosis and under chemotherapy using up to 100cc peripheral blood. After separation for CD4+/CD8+ cells, T cells were activated with anti-CD3/CD28 beads. The lentiviral vector encoded the anti-CD19 single-chain variable fragment, 4-1BB (CD137) co-stimulation and T cell receptor (TCR) zeta chain. The whole process including separation of T cells, activation, transduction and cultivation was performed in a closed and fully automated system. Despite a broad variety in cellular composition including high blast counts, low cell numbers and a rather exhausted phenotype in the starting fraction, a robust T-cell composition was achieved at day five after activation with a mean of 63% CD4+ and 37% CD8+ T cells and a transduction rate of up to 38 %. The vast majority of CAR T cells were of a Tcm (47%) and Tscm (44%) phenotype leading to a strong proliferative potential of more than 100-fold expansion. In addition, a reduced sensitivity to inhibitory signals was documented (programmed cell death protein 1 (PD-1) expression ≤10%). CAR T cells showed effective cytotoxic functionality when co-cultured with CD19+ target cells with only little background of the un-transduced control. At an effector to target ratio of 5:1 up to 80 % of the CD19+ target cells were killed. In addition, a significant release of Interferon gamma (IFN-ɣ), Tumor necrosis factor (TNF-α) and Interleukin-2 (IL-2) was detected upon recognition of the target cell lines, confirming a strong and target-specific Th1 response. In conclusion, generation of CAR T cells from small pediatric blood samples was feasible in a closed GMP-compatible fully automated system. Despite variety of cell numbers, cellular composition and T cell phenotype in the starting sample, a uniform T cell product of Tcm and Tscm could be produced with a balanced CD4 / CD8 ratio leading to high expansion potential and functionality of the T cell graft. Disclosures Blaeschke: Miltenyi Biotec GmbH: Other: Miltenyi Biotec GmbH provided reagents free of charge.. Kaiser:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment.

Description: Unrelated donors are commonly used for hematopoietic stem cell transplants, but graft-versus-host disease (GVHD) is a major problem. We investigated whether transplantation of CD34+ or CD133+ enriched stem cells with add-back of ten million T-cells per kg from unrelated donors would prevent acute GVHD in pediatric patients in combination with pharmacologic immunosuppression. Eighteen patients (1 CML in second chronic phase, 2 MDS, 4 ALL in CR1, 4 ALL in CR2, 1 JMML, 1 AML in CR1, 3 AML in CR2, 1 Wiskott-Aldrich syndrome and 1 NHL in CR2) were transplanted with G-CSF mobilized peripheral blood stem cells (PBSC) from HLA-matched unrelated donors (n = 18). Median of age was 8.9 years (0.5 to 18 years). Conditioning regimens were performed according to national therapy protocol guidelines. On the day of transplant patients received a median of 13.5 (4.5 to 30.0 x 106) CD34+ or CD133+ enriched stem cells and an aliquot of unmanipulated PBSC containing 10 x 106 T-cells per kg. GVHD prophylaxis consisted of cyclosporine A (CSA) and short course methotrexate (MTX) on day +1, +3 and +6. Engraftment was rapid with a median of 19.6 days in sixteen patients. Two patients failed to engraft at first. However, full donor chimerism and stable engraftment was achieved in both patients after cessation of CSA treatment and an additional stem cell boost without any reconditioning. One patient with ALL developed acute GVHD grade III (skin and gut) after cessation of CSA treatment, but responded well to treatment with CSA and steroids. None of the other 17 patients developed acute GVHD 〉 grade I. Thirteen are alive and well with a median follow-up of 578 days (101 to 1095 days). Three patients died of severe infectious complications and two due to relapse (JMML, AML). Compared to a historical control group of patients transplanted with highly purified CD34+ selected cells, the group with add-back of 10 x 106 T-cells per kg showed significantly higher T-cell counts (p=0.008, Wilcoxon Rank sum test) on day 90 after transplantation with a median number of six T-cells/μl in the control group and a median number of 294 T-cells/μl in the study group. We conclude that add-back of 10 x 106 T-cells per kg in combination with CSA and short course MTX improves T-cell recovery and appears to be a safe T-cell dosage regarding acute GVHD in the setting of allogeneic peripheral blood stem cell transplantation from unrelated donors.

Description: We report the results of a pilot study with a clofarabine containing conditioning regimen for allogeneic stem cell transplantation. Several studies have already shown the effectivity of clofarabine in adult patients and children with acute leukemias, resistant to standard treatment protocols. Thus, we replaced fludarabine in a melphalan based myeloablative regimen by clofarabine for pediatric patients with refractory disease after standard treatment (n=7) or after previous transplantation (n=3). A total of 10 pediatric patients with acute lymphatic leukemia (non-remission (NR) 1=3, NR 2=3), acute myeloic leukemia, (NR 1=2) and metastatic rhabdomyosarcoma/neuroblastoma (partial remission (PR) =2) received clofarabine 4×50mg/m2 (day -8 to -5), thiotepa 10mg/kg (day -4), melphalan 2×70mg/m2 (day -3 to -2) and OKT3 (0, 1 mg/kg day -8 to +14; n=9) or ATG (10mg/kg; n=1), followed by infusion of full haplotype mismatched peripheral stem cells (n= 9) or bone marrow from matched unrelated donors (n=1). In mismatched family donors, depletion of T and B cells was carried out with CD3/CD19 coated magnetic microbeads and the CliniMACS® device. A median number of 16×106/kg stem cells (5.6–28) with 90 000/kg residual T cells was infused. Primary engraftment was observed in 9/10 patients. One patient experienced graft rejection but was successfully retransplanted with stem cells from a second parental donor. Thus, sustained engraftment could be obtained in all patients. Median time to ANC 〉500/μl with G-CSF stimulation was 10 days (9–11). Independence from platelet transfusion was reached after 9 days. Median T cell count at day 100 was 138 cells/μl (n=5). 7/10 patients are disease free with a median follow up of 100 days. Single cause of death was relapse of the underlying disease (median time to relapse: 90 days). The regimen was well tolerated without severe side effects. No TRM and no toxicity grade 4 according to NCI-CTC grading system occurred apart from mucositis. The profile of toxic side effects was as follows: gastrointestinal: diarrhea grade 0–2 (n=10), stomatitis grade 3–4 (n=10); skin: none; pulmonary: hypoxia and pneumonitis grade 3 in 2 and 1 patient, respectively; cardiac: none; hepatic: GOT/GPT or bilirubin elevation grade 1–2 in 4 patients, grade 3 in 6 patients; renal: creatinine (-clearance) grade 1 in 1 patient; neurological: none; infection: grade 2 (n=8), grade 3 (n=2). Conclusions: clofarabine was well tolerated as part of a conditioning regimen for allogeneic transplantation. Compared to our standard regimen with fludarabine, thiotepa, melphalan, the introduction of clofarabine did not result in any unexpected toxicity. Immunosuppressive effects appeared to be similar to those of fludarabine, since primary engraftment was observed in 90%. Further studies are warranted to evaluate, whether clofarabine can contribute to reduce the risk of relapse in patients with refractory disease.

Description: Abstract 3548 T and B cell depleted haploidentical grafts and a melphalan based intensity reduced regimen result in low toxicity and stable event free survival in patients with leukemias in CR1-3. However, patients with active disease or with second or subsequent transplantation show unacceptable relapse rates. In an ongoing study, 36 pediatric patients with acute leukemias and advanced MDS (median age: 11 years) received melphalan (2×70mg/m2), fludarabine (4×40mg/m2) or clofarabine (4×50mg/m2), thiotepa (10mg/kg) and OKT3 (0.1mg/kg). T and B cells were depleted by antiCD3/antiCD19 coated magnetic microbeads, whereas NK cells remained into the grafts (median number= 120×106/kg). Remission status was: CR1-3=18, NR=18, 18/36 already received previous allogeneic transplantations. Relapse rates at 2 years were 20% (CR patients) and 73% (active disease patients or 2nd trp). Thus, we investigated options to reduce the risk of relapse by increasing antileukemic activity of donor NK cells in the grafts in vitro. Over night incubation with Interleukin 15 increased NK activity most effectively (specific lysis at E:T=20:1 against K562: 28% prior to and 71% after stimulation, n=10). After additional IL2 stimulation a 22 fold increase in thymidine uptake indicated proliferation of NK cells (n=5). Due to the profound depletion, no T cell proliferation was detectable. Based on these results, we started a pilot study with ex vivo IL15 stimulated grafts in 4 patients at very high risk (ALL, 3rd relapse, active disease (n=1); ALL 2nd relapse, remission (n=1); AML 1st relapse, active disease (n=2)). All patients received a backbone of unstimulated cells at day 0 to ensure engraftment. Additionally, parts of the grafts were incubated over night, washed four times and afterwards infused at day +1 (median numbers: CD56+CD3- =9.4×106/kg (range 3.7–24.4); CD34=1.5×106/kg; CD14=34×106/kg, CD3=0.01×106/kg). No acute side effects occurred. All patients engrafted within 12 days. 3 patients had acute GvDH grade 0-I, 1 patient had GvHD grade III. Recovery of NK cells was remarkably fast (526 CD56+/μl at day 14 posttransplant versus 256 CD56+/μl in patients without IL15 stimulation (means)). After additional administration of IL2 in vivo (1×106 Units/m2/day s.c.) high NK activity (specific lysis〉90% against K 562, E:T=20:1) was detectable in peripheral blood. Two patients are disease free (day 154 and 674 posttransplant), 2 patients died from relapse (day 56 and 64). Conclusions: ex vivo stimulation with IL15 strongly increases cytotoxic activity of NK cells in T and B cell depleted grafts from haploidentical donors and can counterbalance G-CSF mediated inhibitory effects. Those grafts were infused without any acute side effects and resulted in a fast recovery of functional donor NK cells. Potential interactions with stem cells and stem cell derived NK reconstitution have to be investigated. Further studies have to evaluate if this approach might contribute to reduce relapse rates in high risk patients. Disclosures: No relevant conflicts of interest to declare.

Description: Allogeneic stem cell transplantation is still the curative treatment option for the majority of patients with severe aplastic anemia and refractory cytopenias. However, a HLA-matched donor is not available for all patients. Alternative donor transplantation has been an experimental treatment option, limited by high rejection rates and transplant related mortality. We performed a prospective clinical trial to evaluate the safety and feasibility of haploidentical stem cell transplantation, since haploidentical family members are always available donors. We investigated a cohort of 10 pediatric patients with severe aplastic anemia or myelodysplatic syndrome transplanted (refractory cytopenia) with T-cell depleted grafts between 2004 and 2011. 7 patients had myelodyslastic syndrome with refractory cytopenia (MDS-RC), 3 had severe aplastic anemias (SAA) refractory to immunosuppresive treatment. 3 patients received a 2nd SCT after rejecting the graft from matched donors. Median age was 11.4 years. Standard conditioning regimen consisted of Fludarabin 3-4x40mg/m2, Thiotepa 1-3x5mg/kg, Melphalan 2x70mg/m2 (n=8) and serotherapy using OKT3 (n=5) and ATG (n=5). 8 patients received additional total lymphoid irradiation (TLI 7 Gy) to prevent graft rejection. In vitro graft manipulation was carried out by direct depletion using antiCD3/19 magnetic microbeads. A median number of 10.1x106 CD34+ progenitor cells and 27x103 T-cells/kg body weight (BW) were transfused. Pharmacological GvHD prophylaxis (graft vs. host disease) was carried out with Mycofenolate until day 60, if residual T-cells in the graft exceeded 25000/kg BW. Primary engraftment occurred in all patients Median time to reach 500/µl neutrophiles was 9 days (9-11). Independence from platelet substitution was reached after 13 days (8-16). Three patients rejected the graft later on. 6/10 patients had no signs of acute GvHD or GvHD grade I, 2 patients had GvHD grade II. TRM at day +100 and after 1 year was 0% and 20%, respectively. Event free survival (EFS) at 3 years was 80%. Conclusions Haploidentical SCT with T-cell depleted grafts is a therapeutic option for refractory cytopenias and severe aplastic anemia after nonresponse to immunosuppressive treatment if no HLA-matched donor is available. Recovery of neutrophiles and platelets were fast and TRM was low, even if retransplantation was necessary. Since spontaneous outcome of these conditions are poor, alternative donor SCT is a realistic option for these patients. Disclosures: No relevant conflicts of interest to declare.