Matthias Eyrich

Haemopoietic stem-cell transplantation with antibody-based minimal-intensity conditioning: a phase 1/2 study

BACKGROUND Stem-cell transplantation can cure primary immunodeficiencies. However, in patients with pre-existing organ toxicity, patients younger than 1 year, and those with DNA or telomere repair disorders, chemotherapy-based conditioning is poorly tolerated and results in major morbidity and mortality. We tested a novel antibody-based minimal-intensity conditioning (MIC) regimen to assess whether this approach allowed curative donor stem-cell engraftment without non-haemopoietic toxicity. METHODS 16 high-risk patients underwent stem-cell transplantation for primary immunodeficiencies with an MIC regimen consisting of two rat anti-CD45 monoclonal antibodies YTH 24.5 and YTH 54.12 for myelosuppression, and alemtuzumab (anti-CD52) and fludarabine, and low dose cyclophosphamide for immunosuppression. Donors were matched siblings (n=5), and matched (9) and mismatched (2) unrelated donors. FINDINGS Antibody-based conditioning was well tolerated, with only two cases of grade 3 and no grade 4 toxicity. Rates of clinically significant acute (n=6, 36%) and chronic graft-versus-host disease (GVHD) (n=5, 31%) were acceptable. 15 of 16 patients (94%) engrafted, of whom 11 (69%) achieved full or high-level mixed chimerism in both lymphoid and myeloid lineages, and three achieved engraftment in the T-lymphoid lineage only. One patient needed retransplantation. At a median of 40 months post-transplant, 13 of 16 patients (81%) in this high-risk cohort were alive and cured from their underlying disease. INTERPRETATION Monoclonal antibody-based conditioning seems well tolerated and can achieve curative engraftment even in patients with severe organ toxicity or DNA repair defects, or both. This novel approach represents a shift from the paradigm that intensive chemotherapy or radiotherapy, or both, is needed for donor stem-cell engraftment. This antibody-based conditioning regimen may reduce toxicity and late effects and enable SCT in virtually any primary immunodeficiency patient with a matched donor. FUNDING None.

Risk of complications during hematopoietic stem cell collection in pediatric sibling donors: a prospective European Group for Blood and Marrow Transplantation Pediatric Diseases Working Party study.

We investigated prospectively factors influencing the safety of hematopoietic stem cell (HSC) collection in 453 pediatric donors. The children in the study donated either BM or peripheral blood stem cells (PBSCs) according to center policy. A large variability in approach to donor issues was observed between the participating centers. Significant differences were observed between BM and PBSC donors regarding pain, blood allotransfusion, duration of hospital stay, and iron supplementation; however, differences between the groups undergoing BM vs PBSC donation preclude direct risk comparisons between the 2 procedures. The most common adverse event was pain, reported mainly by older children after BM harvest, but also observed after central venous catheter (CVC) placement for PBSC collection. With regard to severe adverse events, one patient (0.7%) developed a pneumothorax with hydrothorax after CVC placement for PBSC collection. The risk of allotransfusion after BM harvest was associated with a donor age of < 4 years and a BM harvest volume of > 20 mL/kg. Children < 4 years were at higher risk than older children for allotransfusion after BM harvest and there was a higher risk of complications from CVC placement before apheresis. We conclude that PBSC and BM collection are safe procedures in children.

Immune function in children under chemotherapy for standard risk acute lymphoblastic leukaemia – a prospective study of 20 paediatric patients

Multidrug chemotherapy is a highly effective treatment for paediatric acute lymphoblastic leukaemia (ALL), but at the same time compromises immunity of patients. Immune function in a homogenous cohort of 20 children with standard‐ and intermediate‐risk ALL was analysed by immunophenotyping, intracellular cytokine staining, assessment of serum cytokine concentrations, T‐cell receptor (TCR) repertoire diversity and thymic function. B‐cells were most severely affected by chemotherapy, rapidly declined under induction and did not recover until the cessation of maintenance therapy. This recovery was paralleled by a relative increase in naive IgM+IgD+CD27− B‐cells, indicating de novo B‐cell generation as the major pathway for B‐cell reconstitution. T‐ and Natural Killer‐cells were less severely affected. Although numerically diminished by chemotherapy, they had partially recovered at the end of induction. Interestingly, CD4:CD8 ratio, distribution of naive versus memory T‐cells, cytokine production, TCR‐repertoire complexity and thymic function were all only marginally affected by chemotherapy. Patients receiving dexamethasone had significantly less IFNγ+ T‐cells than those receiving prednisone. Our data show that during chemotherapy in standard‐ and intermediate‐risk paediatric ALL patients the T‐cell system remains relatively well preserved. Future studies will show if this effect can be exploited for inclusion of immunotherapy in standard ALL treatment protocols.

Risks of mortality in children admitted to the paediatric intensive care unit after haematopoietic stem cell transplantation

Summary. The risk of mortality in children admitted to the paediatric intensive care unit (PICU) after haematopoietic stem cell transplantation is felt to be very high. Life‐threatening complications leading to PICU admission are due to organ toxicity caused by conditioning regimes and graft‐versus‐host disease (GVHD), systemic infections and other organ dysfunctions. Data collected between October 1998 and December 2001 of paediatric patients undergoing haematopoietic stem cell transplantation and thereafter admitted to the PICU were retrospectively analysed in order to reveal the possible causes and risk factors related to death while in the PICU. Twenty‐six PICU admissions were recorded. We found a mortality rate of 57·7% and a 6‐month survival rate of 23·1%. Univariate analysis identified an oncological paediatric risk of mortality (O‐PRISM) score above 10 points, sustained renal failure and a failed negative fluid balance as significant predictors to non‐survival.

Activated memory B cells may function as antigen‐presenting cells in the joints of children with juvenile idiopathic arthritis

OBJECTIVE B cells impact the perpetuation of chronic inflammatory or autoimmune diseases in multiple ways. A role of B cells as antigen-presenting cells (APCs) in the pathogenesis of chronic arthritis in humans has been suggested; however, as of yet the presence of such B cells at the site of inflammation has not been demonstrated. This study was undertaken to investigate whether synovial B cells in patients with juvenile idiopathic arthritis (JIA) might display features of APCs. METHODS The frequency, phenotype, and immunoglobulin repertoire of synovial B cells were studied by flow cytometry and single-cell polymerase chain reaction (PCR). Cytokine expression by B cells was analyzed by real-time PCR, and interaction between B cells and T cells was investigated in a mixed lymphocyte culture. RESULTS CD27+IgD- and CD27-IgD- B cells accumulated in the joints of JIA patients and displayed an activated phenotype. Both B cell subsets expressed hypermutated and class-switched immunoglobulins, indicators of memory B cells. The accumulating memory B cells expressed the costimulatory molecules CD80/CD86 and showed a higher capacity to activate allogeneic T cells and prime a Th1 phenotype than their peripheral blood counterparts. CONCLUSION Activated immunoglobulin class-switched CD27- and CD27+ memory B cells, indicating a phenotype of APCs with expression of costimulatory molecules, accumulate in the joints of patients with JIA and might be involved in the amplification of pathogenic T cell activation. These findings provide evidence that B cells play an antibody-independent immunopathologic role in human chronic inflammatory arthritis of childhood.

Age-related changes in intracellular cytokine expression in healthy children

Cytokine production by human lymphocytes from healthy children (ages 0-18 years) was assessed using a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. To establish valid cytokine values for intracellular cytokine expression in healthy children in the different age groups, we measured 117 samples after 24 h in vitro stimulation with PMA, ionomycin and brefeldin followed by staining with intracellular anti-cytokine and surface antibodies. We found decreasing IL-2 expression, increasing IFN-gamma and TNF-alpha production and stable IL-4, Ki67 and TGFb levels with advancing age. The cytokines were mainly produced by memory T-cells. Apart from age, there was a differential expression in boys and girls: boys (< 6 years) produce significantly more IL-2 (p < 0,04), while girls > 12 years produce more IFNg than boys of the same age (p < 0.05). This systematic analysis of cytokine profiles during childhood allows a better understanding of immune maturation and will contribute significantly to the interpretation of cytokine data from children with pathological conditions.

Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.

Immune response of human propagated gammadelta-T-cells to neuroblastoma recommend the Vdelta1+ subset for gammadelta-T-cell-based immunotherapy.

Human peripheral γδ-T-cells are able to induce cytolysis of neuroblastoma (Nb) tumor cells. Besides innate effector functions against infected cells and tumors, γδ-T-cells are involved in T-helper 1/T-helper 2 (TH1/TH2) differentiation of αβ-T-cells. However, as different γδ-T-cell subsets vary considerably in their functional properties, the aim of the present study was to define repertoires of cytokines, chemokines, and angiogenic factors of in vitro expanded Vδ1+ and Vδ2+ T cells in response to Nb. After short-term culture, both subsets released TH1 [interleukin (IL)-2, interferon (IFN)-γ, IL-12, tumor necrosis factor (TNF)-α, TNF-β)] and TH2 cytokines (IL-4, -5, -6, -10, -13, Vδ1 also transforming growth factor (TGF)-β, chemokines (I-309, monocyte chemotactic protein (MCP)-1-3, regulated upon activation, normal T-cell expressed and secreted), ILs (IL-1, -8, -15), cytokines (leptin) as well as angiogenic growth factors [angiogenin (ANG), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), Insulin-like growth factor (IGF)-I]. These molecules were expressed at higher levels in Vδ2+ than Vδ1+ T cells. Nb challenge changed protein expression. TH2 cytokine and IFN-γ release was blocked in both γδ-T-cell subsets. In Vδ2+ γδ-T-cells, TH1 cytokines were down-regulated and tumor growth-promoting factors (ANG, VEGF, EGF, and IGF-I) were strongly up-regulated. In contrast, Vδ1+ γδ-T-cells stopped the release of tumor-supportive factors and tolerogenic TGF-β, and strongly up-regulated TNF-α, TNF-β, MCP-1 and -2 and maintained their IL-2 production. In summary, our data show that after being challenged with Nb cells, propagated Vδ1+rather than Vδ2+ T cells support antitumor responses by secretion of proinflammatory cytokines. Furthermore, in contrast to other cell types, Vδ1+ T cells do not sustain a growth-promoting or tolerogenic microenvironment. These data make Vδ1+ T cells an ideal candidate for upcoming immunotherapy trials in Nb.

Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells

BACKGROUND AND AIMS One of the major challenges of dendritic cell (DC) vaccination is the establishment of harmonized DC production protocols. Here, we report the transfer and validation of a successfully used open DC manufacturing method into a closed system, good manufacturing practice (GMP)-compatible protocol. METHODS All production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated. RESULTS Tumor lysate was characterized by histology, mechanically homogenized and avitalized. This preparation yielded a median of 58 ± 21 μg protein per milligram of tumor tissue. Avitality was determined by trypan blue staining and confirmed in an adenosine triphosphate release assay. Patient monocytes were isolated by elutriation or CD14 selection, which yielded equivalent results. DCs were subsequently differentiated in Teflon bags for an optimum of 7 days in CellGro medium supplemented with interleukin (IL)-4 and granulocyte macrophage colony stimulating factor and then matured for 48 h in tumor necrosis factor-α and IL-1ß after pulsing with tumor lysate. This protocol resulted in robust and reproducible upregulation of DC maturation markers such as cluster of differentiation (CD)80, CD83, CD86, human leukocyte antigen-DR and DC-SIGN. Functionality of these DCs was shown by directed migration toward C-C motif chemokine ligand 19/21, positive T-cell stimulatory capacity and the ability to prime antigen-specific T cells from naive CD8(+) T cells. Phenotype stability, vitality and functionality of DCs after cryopreservation, thawing and washing showed no significant loss of function. Comparison of clinical data from 146 patients having received vaccinations with plate-adherence versus GMP-grade DCs showed no inferiority of the latter. CONCLUSIONS Our robust, validated and approved protocol for DC manufacturing forms the basis for a harmonized procedure to produce cancer vaccines, which paves the way for larger multi-center clinical trials.

Transient loss of consciousness in pediatric recipients of dimethylsulfoxide (DMSO)-cryopreserved peripheral blood stem cells independent of morphine co-medication.

Toxicity related to the infusion of dimethylsulfoxide-cryopreserved peripheral blood stem cells (DMSO-PBSC) manifests mostly as cardiovascular side effects. Neurotoxicity1 including transient global amnesia,2,3 seizures,4,5 and stroke6,7 has been reported as a rare complication primarily in adults. In children, data8 are sparse. In light of a recent report implicating morphine co-medication as a major contributing factor,8 we evaluated retrospectively our own data base, including all infusions of DMSO-PBSC applied in our pediatric center between January 1st 2002 and December 31st 2008. We report on 2 incidences of transient loss of consciousness following 131 infusions of DMSO-PBSC.