Matthias Pfeiffer

Depletion of T-cell receptor alpha/beta and CD19 positive cells from apheresis products with the CliniMACS device

BACKGROUND AIMSnThe CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαβ(+)) and CD19 positive (CD19(+)) cells from apheresis products.nnnMETHODSnInvestigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2-10.4)xa0× 10(10) mononuclear cells were used with a median 358 (92-1432)xa0× 10(6) CD34(+) cells. There were 24.8% (6.1-45.7%) median TCRαβ(+) cells and 4.4% (1.2-11.7%) median B cells in the apheresis product.nnnRESULTSnAfter depletion, a median 0.00097% (0.00025-0.0048%) of TCRαβ(+) cells could be detected, and B cells, as determined as CD20(+) cells, were reduced to 0.0072% (0.0008-0.072%). TCRαβ(+) cells were depleted by log 4.7 (3.8-5.5), and B cells were depleted by log 4.1 (3.0-4.7). Recovery of mononuclear cells was 55% (33-77%), and recovery of CD34(+) cells was 73% (43-98%). Recovery of CD56(+)/3(-) natural killer cells was 80% (35-142%), recovery of TCR gamma/delta positive (TCRγδ(+)) T cells was 83% (39-173%) and recovery of CD14(+) cells was 79% (22-141%). Viability of cells was 98% (93-99%) after separation (allxa0values median).nnnCONCLUSIONSnProfound depletion of TCRαβ(+) T cells can be achieved with the CliniMACS system. Recovery of CD34(+) stem cells is in the same range than after CD34(+) enrichment and CD3/CD19 depletion. Transplantation with >4xa0× 10(6) CD34(+) cells/kg can be performed for every patient with 1-5xa0× 10(4) TCRαβ(+) cells/kg and about 5-10xa0× 10(6) TCRγδ(+) cells/kg with two rounds of apheresis.

Selective induction of apoptosis in leukemic B-lymphoid cells by a CD19-specific TRAIL fusion protein.

Although the treatment outcome of lymphoid malignancies has improved in recent years by the introduction of transplantation and antibody-based therapeutics, relapse remains a major problem. Therefore, new therapeutic options are urgently needed. One promising approach is the selective activation of apoptosis in tumor cells by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study investigated the pro-apoptotic potential of a novel TRAIL fusion protein designated scFvCD19:sTRAIL, consisting of a CD19-specific single-chain Fv antibody fragment (scFv) fused to the soluble extracellular domain of TRAIL (sTRAIL). Potent apoptosis was induced by scFvCD19:sTRAIL in several CD19-positive tumor cell lines, whereas normal blood cells remained unaffected. In mixed culture experiments, selective binding of scFvCD19:sTRAIL to CD19-positive cells resulted in strong induction of apoptosis in CD19-negative bystander tumor cells. Simultaneous treatment of CD19-positive cell lines with scFvCD19:sTRAIL and valproic acid (VPA) or Cyclosporin A induced strongly synergistic apoptosis. Treatment of patient-derived acute B-lymphoblastic leukemia (B-ALL) and chronic B-lymphocytic leukemia (B-CLL) cells resulted in strong tumoricidal activity that was further enhanced by combination with VPA. In addition, scFvCD19:sTRAIL prevented engraftment of human Nalm-6 cells in xenotransplanted NOD/Scid mice. The pre-clinical data presented here warrant further investigation of scFvCD19:sTRAIL as a potential new therapeutic agent for CD19-positive B-lineage malignancies.

Intensity of HLA class I expression and KIR-mismatch determine NK-cell mediated lysis of leukaemic blasts from children with acute lymphatic leukaemia

The impact of human leucocyte antigen (HLA) class I expression intensity and killer‐cell immunoglobulin‐like receptor (KIR)‐mismatch was investigated on natural killer (NK)‐cell mediated lysis of B‐lineage leukaemic blasts from 21 paediatric patients in vitro. Blast susceptibility to standardised NK‐activity and HLA‐expression differed widely. A clear association between HLA‐molecules/cell (range 442u2003000–13u2003000) and specific lysis (mean 59%, range 13–98%) was observed (r2u2003=u20030·68). A compound model incorporating HLA‐expression and KIR‐ligand‐mismatch provided an even stronger association (r2u2003=u20030·87), whereas KIR‐ligand‐mismatch alone was not significant. Assessment of these factors might identify patients who could benefit from NK‐mediated graft‐versus‐leukaemia effects after mismatched stem cell transplantation.

High Proportion of Leukemic Stem Cells at Diagnosis Is Correlated with Unfavorable Prognosis in Childhood Acute Myeloid Leukemia

In acute myeloid leukemia (AML), the leukemia-initiating cell is found within the CD34+/CD38− cell compartment. Over the last years evidence grew that AML is initiated and propagated by leukemic stem cells (LSCs). Conceivably, these most immature leukemia cells are more resistant to therapy and subsequently initiate relapse. The authors studied 17 patients with childhood AML treated according to the AML-BFM 98/04 protocol. At diagnosis, the authors determined the characteristic immunophenotype of the leukemic cells by flow cytometry and investigated the expression of CD34, CD38, and CD45 to define a population of immunophenotypically immature cells (CD34+/CD38−/CD45−/low) enriched for LSCs in many cases of AML. The authors compared the fraction of this population of all myeloid cells at diagnosis with event-free survival. Kaplan-Meier analysis revealed significant higher event free survival of patients with low CD34+/CD38−/CD45−/low cell proportion (<0.68%) compared to patients with high burden of this population (>0.83%; log-rank P < .04). This correlation was not found for the total number of CD34+ cells. This is the first study to show that a higher proportion of immature CD34+/CD38−/CD45−/low blasts at diagnosis correlates with unfavorable prognosis in childhood AML. The results suggest that a large CD34+/CD38−/CD45−/low population reflects a higher fraction of LSCs, leading to increased chemotherapy resistance and elevated relapse rate. Thus the initial frequency of CD34+/CD38−/CD45−/low cells may serve as a prognostic marker in pediatric AML. Future treatment in childhood AML should specifically target this immature population as well as the mature blast population.

Determination of residual T- and B-cell content after immunomagnetic depletion: proposal for flow cytometric analysis and results from 103 separations

BACKGROUNDnT- and B-cell depletion of apheresis products is an attractive alternative to standard stem cell enrichment in haplo-identical transplantation. Thorough T- and B-cell depletion is necessary for prevention of acute GvHD and T-cell depletion-associated lymphoproliferative disorders. However, the large number of non-T and -B cells in the graft requires special protocols for the determination of extremely low frequencies of residual T cells.nnnMETHODSnApheresis products from healthy donors were T- and B-cell depleted by the CliniMACS system using CD3 and CD19 Ab reagents and the LS tubing set. The recovery of cells and degree of depletion were determined. A four-color multigating strategy was used for enumeration of residual T and B cells.nnnRESULTSnOne-hundred and three separations were performed, with a mean cell recovery of 38+/-12%, CD34 recovery of 61+/-16% and CD56 recovery of 63+/-33%. T and B cells were depleted by log 4.15+/-0.46 and log 3.64+/-0.63, respectively. Four-color multigating flow cytometry allowed the detection of single T cells.nnnDISCUSSIONnCombined T- and B-cell depletion is a feasible method for obtaining stem cell grafts with acceptable stem cell recovery, profound T- and B-cell depletion and a very high amount of NK cells and monocytes. However, analysis of residual T cells is challenging and requires special protocols.

IL-15-stimulated CD3/CD19-depleted stem-cell boosts in relapsed pediatric patients after haploidentical SCT

IL-15-stimulated CD3/CD19-depleted stem-cell boosts in relapsed pediatric patients after haploidentical SCT

Nutritional immunology: function of natural killer cells and their modulation by resveratrol for cancer prevention and treatment

Natural killer (NK) cells as part of the innate immune system represent the first line of defence against (virus-) infected and malignantly transformed cells. The emerging field of nutritional immunology focuses on compounds featuring immune-modulating activities in particular on NK cells, which e.g. can be exploited for cancer prevention and treatment. The plant-based nutrition resveratrol is a ternary hydroxylated stilbene, which is present in many foods and beverages, respectively. In humans it comprises a large variety of distinct biological activities. Interestingly, resveratrol strongly modulates the immune response including the activity of NK cells. This review will give an overview on NK cell functions and summarize the resveratrol-mediated modulation thereof.

Influence of histone deacetylase inhibitors and DNA-methyltransferase inhibitors on the NK cell-mediated lysis of pediatric B-lineage leukemia

Epigenetic drugs like histone deacetylase inhibitors (HDACi) and DNA-methyltransferase inhibitors (DNMTi) have been shown to be effective against a variety of tumor entities. Among different molecular anticancer activities of epigenetic active substances, up-regulation of natural killer (NK) cell ligands was described to contribute to an enhanced NK cell-mediated killing of tumor cell lines. So far, no data is available on this effect in childhood acute lymphoblastic leukemia. We investigated the effect of two HDACi [vorinostat, valproic acid (VPA)] and two DNMTi (azacytidine, decitabine) on the viability, expression of NK ligands, and NK susceptibility of the pre-B-cell-ALL cell line MHH-CALL-4. Whereas vorinostat, azacytidine, and decitabine directly reduced viability of the cell line, VPA had no direct cytotoxic effect. NKG2D-ligands were expressed only at very low levels and not affected by epigenetic treatment. Higher expression was found for the DNAM-1 ligands with significant up regulation of CD112 after treatment with VPA (pu2009=u20090.02). No significant increase in lysis mediated by resting NK cells could be observed, whereas incubation of target cells with decitabine resulted in a significant increase in lysis mediated by IL-2 activated NK cells (pu2009=u20090.0051, pu2009=u20090.06 for azacytidine). Vorinostat and VPA could increase the lysis by expanded NK cells which was statistically not significant due to high inter-individual variability. Furthermore, HDACi but not DNMTi reduced the NK-mediated lysis of MHH-CALL-4 after incubation of effector cells. In conclusion, there is a synergistic effect between epigenetic drugs and NK cells against MHH-CALL-4 which is not as strong as in other tumor entities. In situations where NK-mediated control of leukemia is assumed or wanted, a sophisticated combination of single epigenetic drugs and ex vivo expanded NK cells is needed to maximize the synergistic effect of both treatment strategies and DNMTIs may be preferred based on the direct inhibitory effect of HDACi on NK cell cytotoxicity.

Siglec‐7 tetramers characterize B‐cell subpopulations and leukemic blasts

Cell surface glycosylation has important regulatory functions in the maturation, act‐ivation, and homeostasis of lymphocytes. The family of human sialic acid‐binding immunoglobulin‐like lectins (siglecs) comprises inhibitory as well as activating receptors intimately involved in the regulation of immune responses. Analyses of the interaction between siglecs and glycans are hampered by the low affinity of this interaction. Therefore, we expressed siglec‐7 in eukaryotic cells, allowing for glycosylation, and oligomerized the protein in analogy to MHC tetramers. Using this tool, flow cytometric analysis of lymphocytes became possible. Sialic acid‐dependent binding of siglec‐7 tetramers was confirmed by glycan array analysis and loss of siglec tetramer binding after neuraminidase treatment of lymphocytes. In contrast to most lymphocyte subpopulations, which showed high siglec‐7 ligand expression, B‐cell subpopulations could be further subdivided according to different siglec‐7 ligand expression levels. We also analyzed blasts from acute lymphoblastic leukemias of the B‐cell lineage as well as the T‐cell lineage, since malignant transformation is often associated with aberrant cell surface glycosylation. While pediatric T‐ALL blasts highly expressed siglec‐7 ligands, siglec‐7 ligands were barely detectable on cALL blasts.

Cytokine serum levels during post-transplant adverse events in 61 pediatric patients after hematopoietic stem cell transplantation

BackgroundVeno-occlusive disease, Graft-versus-Host disease, invasive or localized bacterial, viral and fungal infections are known as adverse events after hematopoietic stem cell transplantation representing the major cause for morbidity and mortality. Detection and differentiation of these adverse events are based on clinical symptoms and routine measurements of laboratory parameters.MethodsTo identify the role of cytokines as a possible complication-marker for adverse events, 61 consecutive pediatric patients with a median age of 7.0xa0years who underwent hematopoietic stem cell transplantation were enrolled in this single-center retrospective study. Interleukin-1 beta (IL-1β), soluble interleukin-2 receptor (sIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and tumor necrosis factor-α serum (TNF-α) levels were regularly assessed after transplantation and during transplantation related adverse events.ResultsVeno-occlusive disease was accompanied by a significant increase in levels of IL-6, IL-8 and TNF-α.Graft-versus-Host disease was associated with a significant increase of IL-10, sIL-2R, IL-6 and TNF-α, depending on the respective stage or grade. Cytokine IL-6 enabled a significant differentiation between sepsis and fungemia, sepsis and viremia, and sepsis and bacteremia. Moreover, cytokine IL-8 enabled a significant differentiation between sepsis and viremia, sepsis and bacteremia, and bacteremia and viremia whereas IL-10 made a distinction between sepsis and viremia possible.ConclusionThe data demonstrate that proinflammatory cytokines might be putative indicators for early detection and differentiation of post-transplant adverse events and may allow prompt and adequate clinical intervention. Prospective clinical trials are needed to evaluate these findings.