Transcriptional Response to Standard AML Drugs Identifies Synergistic Combinations.

Unlike genomic alterations, gene expression profiles have not been widely used to refine cancer therapies. We analyzed transcriptional changes in acute myeloid leukemia (AML) cell lines in response to standard first-line AML drugs cytarabine and daunorubicin by means of RNA sequencing. Those changes were highly cell- and treatment-specific.

A PCR protocol to establish standards for routine mycoplasma testing that by design detects over ninety percent of all known mycoplasma species.

Mycoplasma infection leads to false and non-reproducible scientific data and poses a risk to human health. Despite strict guidelines calling for regular mycoplasma screening, there is no universal and widely established standard procedure. Here, we describe a reliable and cost-effective PCR method that establishes a universal protocol for mycoplasma testing.

Definitive evidence for Club cells as progenitors for mutant Kras/Trp53-deficient lung cancer.

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established.

Tumor-priming converts NK cells to memory-like NK cells.

Fascinating earlier evidence suggests an intrinsic capacity of human natural killer (NK) cells to acquire adaptive immune features in the context of cytomegalovirus (CMV) infection or pro-inflammatory cytokine stimulation. Since the role of memory NK cells in cancer has so far remained elusive and adoptive NK cell transfer in relapsing pediatric acute B cell precursor leukemia (BCP-ALL) patients awaits improvement, we asked the question whether tumor-priming could promote the generation of memory NK cells with enhanced graft-vs.-leukemia (GvL) reactivity.

HLA-DPB1 mismatch alleles represent powerful leukemia rejection antigens in CD4 T-cell immunotherapy after allogeneic stem-cell transplantation.

Refractory or relapsed acute myeloid leukemia (AML) represents a frequent complication after allogeneic hematopoietic stem-cell transplantation (HSCT). We show herein that primary in vitro stimulation of CD45RA-selected CD4 T cells of stem-cell donors with 10/10 HLA-matched AML blasts results in expansion of cytolytic T-lymphocytes (CTL) that almost all recognize HLA-DPB1 mismatch alleles, which clinically occur in up to 80% of donor-patient pairs. Primary AML blasts were found to strongly express HLA-DPB1, whereas fibroblasts and keratinocytes used as surrogate target cells for graft-versus-host disease did express HLA-DPB1 only upon IFN-γ pre-treatment.

Patient-individualized CD8⁺ cytolytic T-cell therapy effectively combats minimal residual leukemia in immunodeficient mice.

Adoptive transfer of donor-derived cytolytic T-lymphocytes (CTL) has evolved as a promising strategy to improve graft-versus-leukemia (GvL) effects in allogeneic hematopoietic stem-cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)-reactive CD8(+) CTL with central and effector memory phenotype in a new allogeneic donor-patient specific humanized mouse model.

Exclusive Transduction of Human CD4+ T Cells upon Systemic Delivery of CD4-Targeted Lentiviral Vectors.

Playing a central role in both innate and adaptive immunity, CD4(+) T cells are a key target for genetic modifications in basic research and immunotherapy. In this article, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4(+) cells by surface engineering. When applied to PBMCs, CD4-LV transduced CD4(+) but not CD4(-) cells.

Nanoparticles and antigen-specific T-cell therapeutics: a comprehensive study on uptake and release.

Aim: T lymphocytes are used as cellular therapeutics in many disease entities including cancer. We investigated the uptake and retention of nanoparticles (NPs) by these nonphagocytic cells.

Materials & Methods: Uptake, release and toxicity of various polymeric NP preparations were analyzed by flow cytometry, confocal laser scanning microscopy and transmission electron microscopy.

Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation.

In the last decade there has been a rapid expansion in clinical trials using mesenchymal stromal cells (MSCs) from a variety of tissues. However, despite similarities in morphology, immunophenotype, and differentiation behavior in vitro, MSCs sourced from distinct tissues do not necessarily have equivalent biological properties. We performed a genome-wide methylation, transcription, and in vivo evaluation of MSCs from human bone marrow (BM), white adipose tissue, umbilical cord, and skin cultured in humanized media.

Both mature KIR+ and immature KIR- NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice.

Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.

Superior in vitro stimulation of human CD8+ T-cells by whole virus versus split virus influenza vaccines.

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains.

Subclinical CNS inflammation as response to a myelin antigen in humanized mice.

Multiple sclerosis is a demyelinating autoimmune disease of the CNS. Its animal model experimental autoimmune encephalomyelitis is commonly induced by active immunization with myelin antigens. To investigate human immune responses against myelin antigens in vivo we established a new subclinical experimental autoimmune encephalomyelitis model in humanized mice.

Engraftment of low numbers of pediatric acute lymphoid and myeloid leukemias into NOD/SCID/IL2Rcγnull mice reflects individual leukemogenecity and highly correlates with clinical outcome.

Although immortalized cell lines have been extensively used to optimize treatment strategies in cancer, the usefulness of such in vitro systems to recapitulate primary disease is limited. Therefore, the design of in vivo models ideally utilizing patient-derived material is of critical importance. In this regard, NOD.

Zebrafish xenografts as a tool for in vivo studies on human cancer.

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish.

Monocytes derived from humanized neonatal NOD/SCID/IL2Rγ(null) mice are phenotypically immature and exhibit functional impairments.

Trials of immune-modulating drugs in septic patients have mostly failed to demonstrate clinical efficacy. Thus, we sought to generate a surrogate model of myelomonocytic lineage differentiation that would potentially allow sepsis induction and preclinical testing of anti-inflammatory drugs. Comparing transplantation of cord blood-derived stem cells in neonatal NOD/SCID/IL2Rγ(null) (neonatal huNSG) mice with transplantation of adult peripheral mobilized stem cells into adult NSG (adult huNSG) recipients, we demonstrate that myelomonocytic lineage differentiation in neonatal huNSG mice is retarded and monocytes are phenotypically immature with respect to HLA-DR expression and the emergence of CD80(+)CD86(+) monocytes.

Human epidermal Langerhans cells replenish skin xenografts and are depleted by alloreactive T cells in vivo.

Epidermal Langerhans cells (LC) are potent APCs surveying the skin. They are crucial regulators of T cell activation in the context of inflammatory skin disease and graft-versus-host disease (GVHD). In contrast to other dendritic cell subtypes, murine LC are able to reconstitute after local depletion without the need of peripheral blood-derived precursors.

Alloreactive and leukemia-reactive T cells are preferentially derived from naive precursors in healthy donors: implications for immunotherapy with memory T cells.

Background: HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft-versus-host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens.

Design And Methods: We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7.

Long-term human CD34+ stem cell-engrafted nonobese diabetic/SCID/IL-2R gamma(null) mice show impaired CD8+ T cell maintenance and a functional arrest of immature NK cells.

Allogeneic hematopoietic stem cell transplantation represents the most effective form of immunotherapy for chemorefractory diseases. However, animal models have been missing that allow evaluation of donor-patient-specific graft-versus-leukemia effects. Thus, we sought to establish a patient-tailored humanized mouse model that would result in long-term engraftment of various lymphocytic lineages and would serve as a donor-specific surrogate.

Donor CD4 T cells convert mixed to full donor T-cell chimerism and replenish the CD52-positive T-cell pool after alemtuzumab-based T-cell-depleted allo-transplantation.

Donor lymphocyte infusions (DLI) are used to resolve mixed T-cell chimerism (TCC) after allo-SCT despite a substantial risk of GVHD. We analyzed the impact of prophylactic CD8-depleted (CD8(depl)) DLI in 20 recipients of anti-CD52 alemtuzumab in vivo T-cell-depleted allografts with declining donor TCC after day +60. A total of 13 patients received CD8(depl) DLI and 7 patients did not.

Selective depletion of alloreactive T lymphocytes using patient-derived nonhematopoietic stimulator cells in allograft engineering.

Background: Selective depletion of alloreactive T cells in vitro results in efficient graft-versus-host disease prophylaxis in allogeneic hematopoietic stem-cell transplantation, but it is accompanied by increased recurrence of leukemia. To spare donor T-cell-mediated graft-versus-leukemia immunity against hematopoiesis-restricted minor histocompatibility (minor-H) antigens, we explored the use of patient-derived nonhematopoietic antigen-presenting cells (APC) as allogeneic stimulators for selective allodepletion in leukemia-reactive donor T-cell lines.

Methods: Primary keratinocytes, dermal fibroblasts, and bone marrow fibroblasts were generated from skin biopsies and diagnostic bone marrow aspirates of acute myeloid leukemia patients in vitro.

Rapid identification and sorting of viable virus-reactive CD4(+) and CD8(+) T cells based on antigen-triggered CD137 expression.

Current methods for the detection and isolation of antigen-specific CD4(+) and CD8(+) T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4(+) and CD8(+) memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen.

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8(+) CD62L((high)+) T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rgamma(null) mice.

Objective: Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation.

Materials And Methods: We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8(+) T cells with human leukocyte antigen (HLA) class I-matched AML blasts in microtiter plates.

Depletion of alloreactive donor T lymphocytes by CD95-mediated activation-induced cell death retains antileukemic, antiviral, and immunoregulatory T cell immunity.

In allogeneic hematopoietic stem cell transplantation (AHSCT) graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect are closely but not invariably linked. Thus, harnessing donor lymphocyte mediated GVL immunity and separating it from GVHD is of particular interest. Based on results obtained in murine models we have explored the CD95-mediated activation-induced cell death (AICD) strategy to selectively deplete alloreactivity in human donor T lymphocytes in vitro.

High-temperature-recorded index gratings in periodically poled lithium niobate.

Holographic index gratings based on a zero-electric-field photorefractive effect are recorded at high temperatures in copper-doped periodically poled lithium niobate crystals. The interplay between the domain structure and the index grating is studied: the fundamental grating with spatial frequency K is strongly suppressed. Pronounced sideband gratings with K(s)=K+sG appear, where G is the domain grating vector and s is an integer number.

Determination of the absorption cross section of dopants in lithium niobate crystals.

A method is presented to acquire the absorption cross sections of dopants in photorefractive lithium niobate crystals utilizing doubly doped samples. The absorption cross section of one dopant must be well known. By illumination with ultraviolet light, electrons are transferred from one centre to the other.