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Acute graft-versus-host disease (aGVHD) is an immune cell‒driven, potentially lethal complication of allogeneic hematopoietic stem cell transplantation affecting diverse organs, including the skin, liver, and gastrointestinal (GI) tract. We applied mass cytometry (CyTOF) to dissect circulating myeloid and lymphoid cells in children with severe (grade III-IV) aGVHD treated with immune suppressive drugs alone (first-line therapy) or in combination with mesenchymal stromal cells (MSCs; second-line therapy). These results were compared with CyTOF data generated in children who underwent transplantation with no aGVHD or age-matched healthy control participants. Onset of aGVHD was associated with the appearance of CD11b+CD163+ myeloid cells in the blood and accumulation in the skin and GI tract. Distinct T-cell populations, including TCRγδ+ cells, expressing activation markers and chemokine receptors guiding homing to the skin and GI tract were found in the same blood samples. CXCR3+ T cells released inflammation-promoting factors after overnight stimulation. These results indicate that lymphoid and myeloid compartments are triggered at aGVHD onset. Immunoglobulin M (IgM) presumably class switched, plasmablasts, and 2 distinct CD11b– dendritic cell subsets were other prominent immune populations found early during the course of aGVHD in patients refractory to both first- and second-line (MSC-based) therapy. In these nonresponding patients, effector and regulatory T cells with skin- or gut-homing receptors also remained proportionally high over time, whereas their frequencies declined in therapy responders. Our results underscore the additive value of high-dimensional immune cell profiling for clinical response evaluation, which may assist timely decision-making in the management of severe aGVHD.

Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days.

In contrast to all other blood and immune cells, T lymphocytes do not develop in the bone marrow (BM), but in the specialized microenvironment provided by the thymus. Similar to the other lineages, however, all T cells arise from multipotent hematopoietic stem cells (HSCs) that reside in the BM. Not all HSCs give rise to T cells; but how many and what kind of developmental checkpoints are located along this intricate differentiation path is the subject of intense research. Traditionally, this process has been studied almost exclusively using mouse cells, but recent advances in immunodeficient mouse models, high-speed cell sorting, lentiviral transduction protocols, and deep sequencing techniques have allowed these questions to be addressed using human cells. Here we review the process of thymic seeding by BM-derived cells and T cell commitment in humans, discussing recent insights into the clonal composition of the thymus and the definition of developmental checkpoints, on the basis of insights from human severe combined immunodeficiency patients.

T cells develop from hematopoietic stem cells in the specialized microenvironment of the thymus. The main transcriptional players of T-cell differentiation such as Notch, Tcf-1, Gata3 and Bcl11b have been identified, but their role and regulation are not yet completely understood. In humans, functional experiments on T-cell development have traditionally been rather difficult to perform, but novel in vitro culture systems and in vivo xenograft models have allowed detailed studies on human T-cell development. Recent work has allowed the use of human severe combined immunodeficiency stem cells to unravel developmental checkpoints for human thymocyte development., Lay abstract: T lymphocytes (often simply termed as T cells) are the main disease-fighting cells in the blood. All blood cells are derived from rare cells in the bone marrow that can self-renew: the blood-forming stem cells. While most types of blood cells develop in the bone marrow from these stem cells, T cells develop in a specialized organ called the thymus. Over the last 20 years, researchers have made tremendous progress in understanding the signals that drive the development of T cells, first in mice, but increasingly also in humans. This is important as in many diseases, T cells and their birth in the thymus play an important role. Better insight into this process therefore directly contributes to better human health.

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate the bone marrow of myeloablative conditioned and/or (lethally) irradiated recipients. To study the repopulating potential of human HSCs, murine models have been developed that rely on the use of immunodeficient mice that allow engraftment of human cells. The NSG xenograft model has emerged as the current standard for this purpose allowing for engraftment and study of human T cells. Here, we describe adaptations to the original NSG xenograft model that can be readily implemented. These adaptations encompass use of adult mice instead of newborns and a short ex vivo culture. This protocol results in robust and reproducible high levels of lympho-myeloid engraftment. Immunization of recipient mice with relevant antigen resulted in specific antibody formation, showing that both T cells and B cells were functional. In addition, bone marrow cells from primary recipients exhibited repopulating ability following transplantation into secondary recipients. Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples. Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.

This thesis describes novel insights in human T-cell development by transplanting human HSPCs in severe immunodeficient NSG mice. First, an in vivo model was optimized to allow engraftment of hematopoietic stem cells derived from human bone marrow. This model was used to study aberrant human T-cell development in severe combined immunodeficiency (SCID) and leukemia. For SCID with known mutations as well as with atypical clinical presentation, stages of developmental arrest were determined. Using cellular barcoding, severe restriction of hematopoietic clonality during T-cell development was observed but the limited number of clones still makes up a very diverse polyclonal T-cell receptor repertoire. The restriction points coincided with arrests in development for SCID. In addition, new mechanisms whereby LMO2 causes aberrant human T-cell development, which eventually could lead to leukemia, were identified. The provided model can further aid in both fundamental studies, optimization of gene therapy approaches and stem cell expansion. Furthermore, it illustrates the need for more in depth understanding of human T-cell development, which could help improvement of T-cell reconstitution after transplantation and might aid in the diagnosis and future treatment of patients with T-ALL. Overall, the xenograft model has proven useful to advance our understanding of human T-cell development.

OBJECTIVE: Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown function: CD56-CD127-, CD56-CD127+, CD56+CD127- and CD56+CD127+. Here we aimed to gain insight into the potential function of these innate IELs in health and disease. DESIGN: We determined the phenotypes, relative abundance and differentiation potential of these innate IEL subsets in duodenal biopsies from controls and patients with CD or patients with refractory CD type II (RCDII). RESULTS: Hierarchical clustering analysis of the expression of 15 natural killer and T cell surface markers showed that innate IELs differed markedly from innate peripheral blood lymphocytes and divided innate IEL subsets into two main branches: a CD127- branch expressing high levels of interleukin (IL) 2/15Rβ but no IL-21R, and a CD127+ branch with the opposite phenotype. While CD was characterised by the contraction of all four innate IEL subsets, a selective expansion of CD56-CD127- and CD56-CD127+ innate IEL was detected in RCDII. In vitro, in the presence of IL-15, CD56-CD127- IEL from controls and patients with CD, but not from patients with RCDII, differentiated into functional natural killer and T cells, the latter largely dependent on notch-signalling. Furthermore, compared with non-coeliac controls, CD56-CD127- IEL from patients with CD expressed more intracellular CD3ε and CD3γ and gave more pronounced T cell differentiation. CONCLUSIONS: Thus, we demonstrate previously unappreciated diversity and plasticity of the innate IEL compartment and its loss of differentiation potential in patients with RCDII.

In contrast to all other blood and immune cells, T lymphocytes do not develop in the bone marrow (BM), but in the specialized microenvironment provided by the thymus. Similar to the other lineages, however, all T cells arise from multipotent hematopoietic stem cells (HSCs) that reside in the BM. Not all HSCs give rise to T cells; but how many and what kind of developmental checkpoints are located along this intricate differentiation path is the subject of intense research. Traditionally, this process has been studied almost exclusively using mouse cells, but recent advances in immunodeficient mouse models, high-speed cell sorting, lentiviral transduction protocols, and deep sequencing techniques have allowed these questions to be addressed using human cells. Here we review the process of thymic seeding by BM-derived cells and T cell commitment in humans, discussing recent insights into the clonal composition of the thymus and the definition of developmental checkpoints, on the basis of insights from human severe combined immunodeficiency patients.

Background Severe combined immunodeficiency (SCID) represents congenital disorders characterized by a deficiency of T cells caused by arrested development in the thymus. Yet the nature of these developmental blocks has remained elusive because of the difficulty of taking thymic biopsy specimens from affected children. Objective We sought to identify the stages of arrest in human T-cell development caused by various major types of SCID. Methods We performed transplantation of SCID CD34 + bone marrow stem/progenitor cells into an optimized NSG xenograft mouse model, followed by detailed phenotypic and molecular characterization using flow cytometry, immunoglobulin and T-cell receptor spectratyping, and deep sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor δ (TRD) loci. Results Arrests in T-cell development caused by mutations in IL-7 receptor α (IL7RA) and IL-2 receptor γ (IL2RG) were observed at the most immature thymocytes much earlier than expected based on gene expression profiling of human thymocyte subsets and studies with corresponding mouse mutants. T-cell receptor rearrangements were functionally required at the CD4 − CD8 − CD7 + CD5 + stage given the developmental block and extent of rearrangements in mice transplanted with Artemis-SCID cells. The xenograft model used is not informative for adenosine deaminase–SCID, whereas hypomorphic mutations lead to less severe arrests in development. Conclusion Transplanting CD34 + stem cells from patients with SCID into a xenograft mouse model provides previously unattainable insight into human T-cell development and functionally identifies the arrest in thymic development caused by several SCID mutations.

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements. [KEYWORDS: Adolescent, Cell Proliferation, Child, Child, Preschool, Cluster Analysis, Female, Gene Expression Regulation, Leukemic, Gene Rearrangement, Genome, Human, Homeodomain Proteins/ genetics/physiology, Humans, Infant, MADS Domain Proteins/ genetics/physiology, Male, Myogenic Regulatory Factors/ genetics/physiology, Nuclear Proteins/ genetics/physiology, Oncogenes, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/etiology/ genetics/pathology, Transcription Factors/ genetics/physiology, Tr

Abstract 9 To identify novel oncogenic pathways in T-cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular cytogenetic analyses. Using unsupervised and supervised analyses, we identified a T-ALL cluster that was associated with an immature immunophenotype (CD1−, CD4−, CD8−), frequent expression of CD34 and co-expression of the myeloid markers CD13/CD33. Patients in this cluster lacked any of the known oncogenic rearrangements, but ectopically expressed MEF2C, which was recently demonstrated as an important transcription factor for T-cell development1. Molecular-cytogenetic analyses including the Chromatine Conformation Capture on Chip (4C) method revealed novel rearrangements of the MEF2C locus at 5q14, rearrangement of transcription factors that target MEF2C (PU.1, NKX2-5, RUNX1) or MEF2C-associated cofactors (NCOA2/GRIP1) in about half of the patients in this cluster. Four out of the 6 rearrangements identified have never been observed before in human cancer. Nearly all of these patients in this cluster could be predicted by the early T-cell precursor (ETP) signature2 using PAM statistics. This indicates that MEF2C may represent the oncogene for ETP T-ALL, an entity that has been associated with poor outcome2. Inhibition of MEF2C in a cell line model system provoked relieve of developmental arrest, indicating that ectopic MEF2C expression blocks T-cell development at an early stage. We demonstrated that MEF2C is a transcriptional regulator for many differentially expressed genes that were associated with the immature cluster including LYL1 and LMO2. Although LYL1 has been suggested as potential oncogene for immature T-ALL cases3, oncogenic rearrangements were never identified in T-ALL cases with immature immunophenotype. Our data therefore imply that high expression of LYL1 (and LMO2) is part of a pathogenic pathway for immature T-ALL that is regulated by the MEF2C oncogene. 1 Stehling-Sun, S., Dade, J., Nutt, S. L., DeKoter, R. P. & Camargo, F. D. Regulation of lymphoid versus myeloid fate ’choice’ by the transcription factor Mef2c. Nat Immunol 10, 289–296, (2009). 2 Coustan-Smith, E. et al. Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia. Lancet Oncol 10, 147–156, (2009). 3 Ferrando, A. A. et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell 1, 75–87 (2002). Disclosures: No relevant conflicts of interest to declare.

Abstract 3139 To identify novel oncogenic pathways in T-cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular cytogenetic analyses. Using unsupervised and supervised analyses, we identified a novel T-ALL entity that was associated with high expression of cell cycle genes and was accordingly denoted as the proliferative cluster. Patient samples in this cluster were devoid of known oncogenic rearrangements, and clustered together with TLX1-rearranged cases in unsupervised cluster analysis suggesting pathogenic mechanisms are conserved between these cases. Alike TLX1-rearranged cases, patients within this cluster were associated with cortical thymic arrest and expressed CD1 along with CD4 and CD8. Bioinformatic analyses identified NKX2-1 as strong outlier gene that was ectopically expressed in most patients samples belonging to this cluster. One patient sample had high expression of the homologous NKX2-2 gene. Molecular-cytogenetic analyses including the Chromatine Conformation Capture on Chip (4C) method revealed various rearrangement variants of the NKX2-1 locus at 14q13 in 6 patients, including inversions to the T-cell receptor alpha/delta locus (TRAD@) at 14q11, an inversion to the IgH@ locus at 14q32 and a translocation to the TRB@ locus at 7q34. A translocation of NKX2-2 to the TRAD@ locus was identified in the case that ectopically expressed NKX2-2. All these rearrangements were novel, and have not been observed before in human cancer. NKX2-1 rearrangements could be validated in an independent T-ALL cohort. Our data strongly imply that NKX2-1/NKX2-2 represent novel oncogenes for human T-ALL. Disclosures: No relevant conflicts of interest to declare.

The paper will deal with new developments on basis of the ideas, laid down in ASME paper 90-GT-180, presented at the Brussels Conference. In this former paper a combination of incinerators and cogen systems was described. New development show, that some of these ideas can also be used in cogen plants, in which all steam is raised and superheated in a waste heat boiler behind a high grade fuel fired gas turbine (natural gas or equivalent). This paper will deal give a description of the new system. A comparison will be made with conventional cogen systems, comprising of a gas turbine, a dual pressure non-fired waste heat boiler and a condensing steam turbine. On basis of a particular case study both the technical and financial performances will be compared with each other.Copyright © 1992 by ASME

In this paper a new process is described to improve the efficiency of power generation in waste incineration plants. It is proposed to superheat the steam in a gas turbine/waste heat boiler combination rather than in the incinerator boiler. Although this is not discussed in this paper the same process may be applied in nuclear power stations where the superheating of steam is also a problem. A global risk-analysis of the addition of equipment is made in this paper. On basis of a case-study the financial advantages are demonstrated.